ASSESSING STATUS OF AND RESISTANCE TO

PHYTOPHTHORA ON

TRUE (ABIES SPP.)

By

KATHLEEN MARY MCKEEVER

A dissertation submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY Department of

DECEMBER 2016

© Copyright by KATHLEEN MARY MCKEEVER, 2016 All Rights Reserved

© Copyright by KATHLEEN MARY MCKEEVER, 2016 All Rights Reserved

To the Faculty of Washington State University:

The members of the Committee appointed to examine the dissertation of KATHLEEN

MARY MCKEEVER find it satisfactory and recommend that it be accepted.

______Gary A Chastagner, Ph.D., Chair

______Dennis A. Johnson, Ph.D.

______Ned B. Klopfenstein, Ph.D.

______Mark Mazzola, Ph.D.

ii

ACKNOWLEDGEMENT

Thank you Dr. Gary Chastagner, my advisor through two degrees and eight years, for sharing with me your incredible wisdom and interminable curiosity, unequivocal patience and diplomacy, and commitment to scholastic and professional achievement. You’ve provided me with the skills to excel as a pathologist, extension educator, critical thinker, and a burgeoning human being. Thanks to my doctoral committee, Dr. Dennis Johnson (WSU), Dr. Mark Mazzola

(USDA ARS), and Dr. Ned Klopfenstein (USDA USFS) for their collaboration, judicious guidance, and thoughtful feedback throughout. This research could never have been completed without the collaboration, selflessness, physical labor, mentorship and kinship of my labmates at

WSU Puyallup Research and Extension Center, Katie Coats, Andree DeBauw, Gil Dermott, Dr.

Marianne Elliott, Dr. Andrea Garfinkel, Dr. Anna Leon, David McLoughlin, Andy McReynolds,

Kathy Riley, Lucy Rollins, Don Sherry, and Carly Thompson. Finally, thank you to Jordan

Bowerman, my husband and the most extraordinarily capable, meticulous, and resourceful person I’ve ever met – the logistic escapades that were encountered in the course of this research were conceivable and achievable through the dedication and ingenuity of this man. You merit credit for this degree too.

iii

ASSESSING STATUS OF AND RESISTANCE TO

PHYTOPHTHORA ROOT ROT ON

TRUE FIR (ABIES SPP.)

Abstract

by Kathleen Mary McKeever, Ph.D. Washington State University December 2016

Chair: Gary A Chastagner

Many fir species (Abies) are susceptible to Phytophthora Root Rot (PRR). Poor drainage and standing water facilitate pathogen survival, proliferation, and spore dispersal. Disease is caused by numerous Phytophthora spp. that constitute regionally-adapted communities. Survey results of diseased Abies in U.S. Christmas farms demonstrated a nearly uniform community profile dominated by P. cambivora on noble fir in the Pacific Northwest, and a more diverse array of Phytophthora species collected from Fraser, Turkish, balsam, and Canaan in the

Great Lakes and Eastern states. It was concluded that Phytophthora community compositions vary in response to host availability, environment, and anthropogenic distribution of infested host material.

Aside from short-term chemical and cultural individual-tree protection, there are few feasible methods for altering the environment to provide perennial disease abatement. An alternative approach is to reform host conduciveness to disease by identifying resistant members of a population through the application of molecular tools. As a precursor to molecular marker

iv development, a multifactorial study was performed to characterize host phenotypes in response to multiple species of Phytophthora under variable environmental conditions. It was demonstrated that P. cinnamomi and P. taxon kelmania cause greater root damage and more mortality than P. pini or P. cambivora and that disease is more severe at warmer temperatures.

Furthermore, it was established that there is a spectrum of resistances among the various species of Abies, ranging from highly susceptible noble and Fraser firs to more resistant Turkish and

Nordmann firs.

Previous observations of PRR-resistant Turkish fir plantings have indicated that the ability to resist disease is not uniform among seed sources. To test variability among Turkish fir families, a collection of 36 seed sources was inoculated with five Phytophthora species under saturated field conditions. Chi square analysis indicated that four seed sources were significantly more susceptible; however, mortality of less than 10% in the most susceptible Turkish fir families in comparison to 100% mortality in Fraser and noble fir suggested that Turkish fir is a

PRR-resistant Abies species. Significantly more disease caused by P. cryptogea in this field study confirmed previous observations of variability in Phytophthora species aggressiveness.

v

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENT ...... iii

ABSTRACT ...... iv

LIST OF FIGURES ...... viii

LIST OF TABLES ...... x

INTRODUCTION ...... 1

Literature Cited ...... 5

CHAPTER ONE: A SURVEY OF PHYTOPHTHORA SPP. ASSOCIATED WITH ABIES IN

U.S. CHRISTMAS TREE FARMS ...... 8

Literature Cited ...... 25

CHAPTER TWO: INTERACTIONS BETWEEN ROOT-ROTTING PHYTOPHTHORA, ABIES,

AND ENVIRONMENT...... 32

Literature Cited ...... 53

CHAPTER THREE: FIELD ASSESSMENT OF TURKISH FIR (ABIES

BORNMUELLERIANA) RESISTANCE TO FIVE ROOT-ROTTING PHYTOPHTHORA

SPECIES ...... 57

Literature Cited ...... 77

APPENDIX

CHAPTER 1 FIGURES AND TABLES ...... 83

vi

CHAPTER 2 FIGURES AND TABLES ...... 87

CHAPTER 3 FIGURES AND TABLES ...... 100

vii

LIST OF FIGURES

Page

CHAPTER ONE

Figure 1. Maximum Likelihood tree using rDNA ITS sequences showing phylogenetic relationships between Phytophthora isolates collected during this survey and reference

Phytophthora sequences obtained from GenBank ...... 83

CHAPTER TWO

Figure 1. Mean lesion sizes produced on noble fir seedling stems seven days post-inoculation with various isolates of four Phytophthora species LIST OF TABLES ...... 87

Figure 2. Relationship between log-transformed area under the disease progress curve (AUDPC) values and root rot rating (expressed as the midpoint value of the percentage range) for each host species...... 88

Figure 3. Relationship between log-transformed area under the disease progress curve (AUDPC) values and seedling dry weights (expressed as the percentage of the control seedlings) for each host species...... 89

Figure 4. Seedling root system dry weights (expressed as the percentage of the control seedling weights) among host species for each Phytophthora species and isolate treatment...... 90

Figure 5. Seedling root system dry weights (expressed as the percentage of the control seedling weights) among Phytophthora species for each host species...... 91

Figure 6. Average root rot severity (expressed as the midpoint values of the percentage range) among Phytophthora species for each host species...... 92

viii

Figure 7. Average root rot severity (expressed as the midpoint value of the percentage range) among host species for each Phytophthora species and isolate treatment...... 93

Figure 8. Disease progress curves displaying mortality over 16 weeks for seven Abies species exposed to three isolates each of four Phytophthora species at two temperatures...... 94

Figure 9. In vitro radial growth (mm/day) of three isolates of each of four species of

Phytophthora at the two temperatures employed in the greenhouse study ...... 95

CHAPTER THREE

Figure 1. Percent mortality of 36 seed sources of Turkish fir, two seed sources of Nordmann fir, and one seed source each of noble and Fraser firs ...... 100

Figure 2. Percent mortality among Turkish fir produced from three provenances...... 101

Figure 3. Mortality of seedlings from seed sources from three elevation categories within Bolu,

Karabük, or Adapazarı Turkish fir provenances...... 102

Figure 4. Percentage of the two-year total seedlings of each Abies host killed by each

Phytophthora spp...... 103

Figure 5. Percentage of two-year total tree mortality caused by each Phytophthora species, irrespective of host species...... 104

Figure 6. Map depicting the southwestern Black Sea and Marmara regions of northwestern

Turkey ...... 105

ix

LIST OF TABLES

Page

CHAPTER ONE

Table 1. Phytophthora spp. isolated from Abies tissue or associated soils at Christmas tree farms in nine U.S. states ...... 84

Table 2. Pathogenicity of Phytophthora spp. found on Abies hosts for the purpose of establishing new disease reports for various locations ...... 86

CHAPTER TWO

Table 1. Bulk custom soil mix prepared by Specialty Soils (Covington, WA) used to pot seedlings for all experiments...... 96

Table 2. Phytophthora species and isolates utilized for virulence testing to obtain the 3 most aggressive isolates of P. cambivora, P. cinnamomi, P. kelmania, and P. pini...... 97

Table 3. Results from Pearson’s Product Moment Correlation and multiple linear regression to compare three response variables ...... 98

Table 4. Average percent mortality of seven Abies spp. after 16 weeks exposure to three isolates each of four Phytophthora species at two temperatures ...... 99

CHAPTER THREE

Table 1. Phytophthora cultures utilized in both years of the field experiment ...... 105

x

DEDICATION

This dissertation is dedicated to my family – the most splendid collection of hearts, minds, wits, and characters that a person could ever be so lucky to have. They have always championed

Jordan’s and mine journeys in the West, sustaining our inanities with interest, enthusiasm, and compassion; and they’ve striven to keep us close in the fabrics of our tribes. Thank you to all of you for allowing us the freedom to pursue our dreams - we are always working our way back to you.

xi

General Introduction.

Phytophthora Root Rot (PRR) is a serious disease of fir (Abies spp.), resulting in substantial losses in the Christmas tree and nursery industries (Chastagner and Benson

2000; McKeever and Chastagner 2016). Disease is favored by abundant soil moisture common with poorly-drained soils or excessive irrigation (Chastagner et al. 1995; Cooley et al. 1985;

Erwin and Ribeiro 1996; Hamm et al. 1984). Above-ground symptoms associated with PRR include resinous stem cankers, cambial lesions with distinct margins, branch flagging, diminished apical leader growth, wilting of succulent spring foliage, foliar chlorosis, and a characteristic reddening of the needles with advanced infection and senescence (Chastagner and

Benson 2000; Chastagner et al. 1995). Phytophthora infections on roots result in a cortical rot characterized by darkened tissue and sloughing root bark. On some host species, root infections can spread through the vascular system leading to sub-rhytidomal stem lesions (Chastagner and

Benson 2000; Cooley et al. 1985). Senescence and mortality can occur from coalescence of cambial necrosis resulting in of the tree and/or loss of water and nutrient acquisition due to reduced capacity of the root system (Chastagner et al. 1990).

Increases in consumer demand for desirable true fir Christmas has resulted in a present-day cut-Christmas tree industry worth more than 300 million U.S. dollars annually

(USDA NASS 2014). As of 2012, more than 300 thousand acres of land were dedicated to

Christmas tree production in the United States. The efforts of cultivating a tree to market maturity (7-10 years) can be undermined in mere months when conditions coincide to favor viability and spread of Phytophthora species. Heavy PRR infestations can impact susceptible

Abies crops at all stages of maturity, often striking at prime market age. Despite the availability of select chemical alternatives and cultural control methods, practical options for reducing PRR

1 losses in Christmas tree farms are scarce, and their utilization may be limited by expense or feasibility of execution (Frampton and Benson 2012).

Although chemical fumigants are available for commercial nursery production, they are unavailable for use in the Christmas tree industry. In Christmas tree where mature (1.5

– 2 m) trees are in need of protection, the approved chemicals only serve to provide interim defense for the immediate growing season and fail to address the larger issues of existing pathogen residency and the inadequate drainage that favors disease development (Frampton and

Benson 2012; Talgø and Chastagner 2013). Extreme susceptibility of Fraser (Abies fraseri

(Pursh) Poir.) and noble (A. procera Rehder) fir often exceeds the practical efficacy of existing control measures available to Christmas tree farmers and has dictated grower abandonment of

Phytophthora-infested land for cultivation of these highly desirable species (McKeever and

Chastagner 2016).

Disease occurs on an array of Abies spp. in the United States and Europe. Fraser fir is susceptible to infection by P. cinnamomi, P. cactorum (Lebert & Cohn) J. Schröt, P. citricola

(Leonian) Emend., P. drechsleri Tucker, and P. plurivora Jung & Burgess (Adams and Bielenin

1988; Benson et al. 1976; Chastagner and Benson 2000; Frampton and Benson 2004; Jung and

Burgess 2009; Kuhlman and Hendrix 1963). Balsam fir (A. balsamea (L.)Mill.) and white fir (A. concolor (Gordon) Lindley ex Hildebrand) have been demonstrated to be hosts for P. cinnamomi, P. citricola, and P. cactorum (Adams and Bielenin 1988; Grand 1985; Kenerley and

Bruck 1981). Nordmann fir (A. nordmanniana (Steven) Spach.) has been infected by P. cinnamomi in the U.S. and also with P. inundata Brasier, Sanch.-Hern., & Kirk in Norway and

P. niederhauserii Abad & Abad in Hungary (Abad et al. 2014; Grand 1985; Talgø et al. 2007).

Noble fir is particularly vulnerable to PRR, with disease reported to be caused by P. cambivora,

2

P. cactorum, P. cryptogea Pethybr. & Laff., P. cinnamomi, P. megasperma Drechsler, P. gonapodyides (Petersen) Buisman, and P. citricola (Adams and Bielenin 1988; Chastagner et al.

1990a; Chastagner et al. 1995; Hamm and Hansen 1982; Talgø et al. 2006). Other true firs that have been shown to be susceptible to a complex of Phytophthora spp. include subalpine fir (A. lasiocarpa (Hooker) Nuttall), silver fir (A. amabilis Douglas ex J. Forbes), Shasta red fir (A. magnifica A. Murray var. shastensis Lemmon), and grand fir (A. grandis (Douglas ex D. Don)

Lindley) (Chastagner et al. 1990a; Hamm and Hansen 1982; Talgø et al. 2007).

Additional Phytophthora spp. that have been implicated in the PRR complex on true fir and Douglas-fir (Pseudotsuga menziesii (Douglas) ex D. Don) include P. pseudotsugae Hamm &

Hans., P. sansomeana Hans. & Reeser, and the yet undescribed Phytophthora taxon referred to as ‘kelmania’ (Abad et al. 2002; Hamm and Hansen 1983; Hansen et al. 2009). The species structures of Phytophthora communities appear to vary regionally in accordance with distribution of hosts and environmental conditions, such as soil moisture and temperature.

Disease management and classifying Abies species as resistant or susceptible can be complicated by the large number of hosts, the variety of different Phytophthora species, and the effect of ambient environment on both pathogen and host.

The reduction of annual revenue and abandonment of otherwise productive fields to PRR is a problem in an industry where large fiscal inputs are dedicated over several years to produce a crop in which returns are earned during only a small retail window. The best way to increase management efficacy for agricultural Abies producers is to tailor management activities to a regional scale in order to avoid the confluence of conditions favorable to PRR. Defining host- pathogen interactions among species of root-rotting Phytophthora and Abies will improve the ability to defend against PRR and bolster options for cultural management. To this end, the

3 studies commenced forth in this paper have been undertaken in an effort to improve the current knowledge base of Phytophthora-Abies interactions by (1) surveying the current prevalence of disease-causing Phytophthora species in U.S. Christmas tree farms, (2) employing a subset of these species in detailed inoculation experiments to assess host resistance to disease under variable ambient conditions, and (3) screening families of putatively resistant Turkish fir (Abies bornmeulleriana) to determine the potential for this species to serve as an alternative to susceptible Abies hosts. The ultimate goals of the completed research are to provide sound advice in pursuit of minimizing the impact of PRR in Christmas tree farms and conifer nurseries, to assist in the preliminary identification of molecular markers that may be associated with host resistance, and to support the successful production and continued economic profitability of associated industries.

4

Literature Cited

Abad, Z. G., Abad, J. A., Creswell, T. 2002. Advances in the integration of morphological and

molecular characterization in Phytophthora genus: The case of P. kelmania and other

putative new species. Phytopathology 92 (6 SUPPL.): S1.

Abad, Z.G., Abad, J.A., Cacciola, S.O., Pane, A., Faedda, R., Moralejo, E., Perez-Sierra,

A., Abad-Campos, P., Alvarez-Bernaola, L.A., Bakonyi, J., Jozsa, A., Herrero, M.L.,

Burgess, T.I., Cunnington, J.H., Smith, I.W., Balci, Y., Blomquist, C., Henricot, B., Denton,

G., Spies, C., McLeod, A., Belbahri, L., Cooke, D., Kageyama, K., Uematsu, S., Kurbetli, I.,

and Degirmenci, K. 2014. Phytophthora niederhauserii sp. nov., a polyphagous species

associated with ornamentals, fruit trees and native in 13 countries. Mycologia 106:

431-447.

Adams, G.C., and Bielenin, A. 1988. First Report of Phytophthora cactorum and P. citricola

causing crown rot of fir species in Michigan. Plant Disease 72: 79.

Benson, D.M., Grand, L.F., Suggs, E.G. 1976. Root rot of Fraser fir caused by Phytophthora

drechsleri. Plant Dis. Rep. 60: 238-240.

Chastagner, G. A., and Benson, D. M. 2000. The Christmas tree: traditions, production, and

diseases. Online. Plant Health Progress doi:10.1094/PHP-2000-1013-01-RV.

Chastagner, G.A., Hamm, P.B., Byther, R. 1990. Symptomology of Phytophthora root and stem

canker disease of noble fir in the Pacific Northwest. Phytopathology 80: 887.

Chastagner, G.A., Hamm, P.B., Riley, K. L. 1995. Symptoms and Phytophthora spp. associated

with root rot of noble fir Christmas trees in the Pacific Northwest. Plant Disease 79: 290-293.

Cooley, S.J., Hamm, P.B., Hansen, E.M. 1985. Management guide to Phytophthora root rot in

5

bareroot conifer nurseries of the Pacific Northwest. USDA Service, Pacific Northwest

Region. 13 pp.

Erwin, D.C. and Ribeiro, O.K. 1996. Phytophthora Diseases Worldwide. St. Paul, Minnesota:

APS Press. 562 pp.

Frampton, J., and Benson, D.M. 2012. Seedling resistance to in the

genus Abies. Annals of Forest Science. Online. DOI 10.1007/s13595-012-0205-4.

Frampton, J. and Benson, D.M. 2004. Phytophthora root rot mortality in Fraser fir seedlings.

HortScience 39(5): 1025-1026.

Grand, L.F., Ed. 1985. North Carolina Plant Disease Index. North Carolina Agric. Res. Serv.

Techn. Bull. 240:1-157.

Hansen, E.M., Wilcox, W.F., Reeser, P.W., Sutton, W. 2009. Phytophthora rosacearum and P.

sansomeana, new species segregated from the Phytophthora megasperma ‘‘complex’’.

Mycologia 101(1): 129-135.

Hamm, P.B., and Hansen, E.M. 1983. Phytophthora pseudotsugae, a new species causing root

rot of Douglas-fir. Canadian Journal of Botany 61(10): 2626-2631.

Hamm, P.B., and Hansen, E.M. 1982. Pathogenicity of Phytophthora species to Pacific

Northwest . European Journal of Forest Pathology 12: 167-174.

Jung, T., and Burgess, T.I. 2009. Re-evaluation of Phytophthora citricola isolates from multiple

woody hosts in Europe and North America reveals a new species, Phytophthora plurivora sp.

nov. Persoonia 22: 95-110.

Kenerley, C.M., and Bruck, R.I. 1981. Phytophthora root rot of balsam fir and Norway spruce in

North Carolina. Plant Disease 65: 614-615.

Kuhlman, E.G., and Hendrix, F.F. Jr. 1963. Phytophthora root rot of Fraser fir. Plant Disease

6

Reporter 47(6): 552-553.

McKeever, K.M. and Chastagner, G.A. 2016. A survey of Phytophthora species associated with

Abies in U.S. Christmas tree farms. Plant Disease 100(6): 1161-1169.

Talgø, V. and Chastagner, G.A. 2013. Phytophthora on Abies spp. JKI Data Sheets – Plant

Diseases and Diagnosis 77. Online. ISSN: 2191-1398, doi: 10.5073/jkidspdd.2013.077.

Talgø, V., Herrero, M.L., Toppe, B., Klemsdal, S.S., Stensvand, A. 2007. Phytophthora root rot

and stem canker found on Nordmann and subalpine fir in Norwegian Christmas tree

. Online. Plant Health Progress. doi: 10.1094/PHP-2007-0119-01-RS.

Talgø, V., Herrero, M., Toppe, B., Klemsdal, S., Stensvand, A. 2006. First Report of root rot and

stem canker caused by Phytophthora cambivora on noble fir () for bough

production in Norway. Plant Disease 90(5): 682.

United States Department of Agriculture, National Agricultural Statistics Service. "2012 Census

of Agriculture." USDA - NASS - Census of Agriculture, issued May 2, 2014. Web. 6 Nov.

2015.

sv1.pdf>.

7

Chapter 1. A Survey of Phytophthora Species Associated with Abies in U.S. Christmas Tree

Farms

Varying host susceptibilities and regional differences in Phytophthora community compositions complicate disease avoidance and management for Christmas tree growers and conifer nurseries. Updated information regarding regional pathogen species prevalence and relative host susceptibilities can facilitate disease prevention by improving site management strategies for Phytophthora suppression. One of the most fundamental methods for determining the scope and impact of any given disease is to gain an understanding of host/pathogen distributions through the detection and sampling of impacted areas.

Much of the previous literature regarding Phytophthora Root Rot (PRR) on Abies is comprised of surveys that were initiated in response to PRR outbreaks in nurseries or farms, and only a few studies have purposely surveyed a number of geographic locations for the purpose of characterizing community structures and regional variation (Hamm and Hansen 1982, 1983;

Kenerley and Bruck 1981; McCain and Scharpf 1986; Shew and Benson 1981). In the

Northwestern U.S., a 1976 survey of conifer nurseries and regeneration sites in Oregon and

Washington established that P. cinnamomi, P. drechsleri, and P. cryptogea were causing mortality of Douglas-fir seedlings and orchard trees, but this study did not survey Abies (Pratt et al. 1976). A survey of 16 Oregon and Washington bareroot conifer nurseries in 1979 yielded P. drechsleri, P. megasperma, P. cryptogea, P. cactorum, and P. cinnamomi, but made no mention of the host species from which these Phytophthora spp. were isolated (Hansen et al. 1979). A

1990 study by Chastagner et al. found P. cambivora, P. cryptogea, P. cinnamomi, and a P. gonapodyides-like isolate in a survey of four noble fir plantations in Oregon (Chastagner et al.

1990a). This study was followed up five years later with a survey of 30 Oregon and Washington

8 noble fir Christmas tree plantations where at least seven Phytophthora spp. were found causing root and stem canker symptoms on 48 trees in 20 of the 30 sampled plantations (Chastagner et al.

1995). This study revealed the dominance of P. cambivora and P. megasperma on noble fir, and also presented evidence of rot caused by P. cactorum, P. citricola, P. cinnamomi, and P. gonapodyides (Chastagner et al. 1995). Grand & Lapp (1974) tracked fir mortality in Christmas tree plantations in western North Carolina during a 10-year period. During this time, Fraser fir was the only host sampled from 14 plantations and P. cinnamomi was the only Phytophthora sp. identified causing PRR (Grand and Lapp 1974). A larger survey performed by Benson & Grand in 2000 surveyed 58 state-wide North Carolina Christmas tree farms and 16 nursery transplant beds, sampling from >1,000 Fraser fir seedlings and older, established trees. Over 90% of the mortality appeared to be caused by P. cinnamomi, but P. cactorum and P. drechsleri were also found (Benson and Grand 2000). In 1988, Adams & Bielenin observed P. cactorum and P. citricola on balsam, noble, white, Fraser, and Douglas-fir seedlings in various conifer nurseries on the lower peninsula of Michigan (Adams and Bielenin 1988). More recently, a general survey of 30 nursery, greenhouse, and landscape sites in Iowa, Michigan, Ohio, and Indiana revealed 13

Phytophthora species affecting a variety of horticultural hosts including Abies, but no details were provided regarding which species of fir were sampled or the identities or frequency of

Phytophthora spp. that were recovered from fir (Leonberger et al. 2013).

One issue that complicates the quality of disseminated research information regarding host species resistance is that greenhouse data indicate variability in host resistance dependent on the Phytophthora species present. Inoculation trials performed at Washington State University

(unpub) found less than 30% damage on Abies spp. such as Turkish, Nordmann, white, and balsam fir caused by P. cambivora and P. pini, which occur commonly in the Pacific Northwest.

9

In contrast, trials performed by researchers at North Carolina State University demonstrated mortality percentages as high as 60-80% in Turkish and Nordmann firs, respectively, and nearly

100% in white and balsam firs when exposed to P. cinnamomi, which is prevalent in southeastern growing regions (Frampton and Benson 2012). For this reason, a clearer understanding of current regional Phytophthora community structures and host reactions to PRR is needed. By clarifying the dynamics of the PRR system, researchers will be better equipped to investigate methods to exploit host resistance for PRR abatement and provide proper recommendations to growers about which host species will be most successful in their local areas. This paper summarizes the results of a survey of root-rotting Phytophthora in U.S.

Christmas tree plantations in order to characterize regional Phytophthora species diversity and observe geographic differences in the susceptibility of various Abies species. Additionally, there was an opportunity to explore the possibility for potential parallels between current pathogen community compositions and an increased diversity in the number of sources providing planting material for the Christmas tree industry.

Throughout the course of the survey, it was acknowledged that several of the Abies-

Phytophthora combinations that were observed were previously unreported in the literature, prompting motivation to complete Koch’s Postulates for each novel interaction in order to provide proof of pathogenicity of these Phytophthora species on their observed hosts and establish new disease reports.

Materials and Methods

Study Sites. Survey efforts were concentrated in five major U.S. Christmas tree production regions during a two-year period from 2012 to 2013. The Pacific Northwestern

(PNW) region included 12 Christmas tree plantations in western Washington, six in western

10

Oregon, and three sites in northern Idaho. The Western region included five sites in northern

California. The Northeastern region was represented by two sites in Connecticut and one in New

York, while the Southeastern region was comprised of four sites in North Carolina. Archived

Phytophthora cultures from Michigan and Wisconsin were included to represent farms in the

Great Lakes region. Cultures from Michigan were collected in 2005 by researchers at Michigan

State University at an undisclosed number of sites in five counties. Cultures from Wisconsin were collected over a three-year period from 2010 to 2013 by Wisconsin Department of

Agriculture, Division of Trade and Consumer Protection (DATCP) personnel from various sites in three counties. In general, samples in Michigan were isolated from the soil surrounding Fraser fir seedlings in a transplant bed, while the Wisconsin collection came directly from the roots of species including Fraser, balsam, and Canaan firs.

Isolation Methods. Diseased trees were recognized by visual detection of common root rot symptoms including flagging lower branches, stunted leader growth, wilting shoots, and chlorotic and necrotic foliage. In many instances, large cinnamon-brown bole lesions could be located by scraping back the bark around dead branches on the lower stems of noble and Fraser firs. Mature (1.5 – 2 m) trees were sampled either by exposing necrotic stem lesions or by uprooting entire trees and exposing rotten roots. One-square-centimeter pieces of woody tissue from the margins of stem and root lesions were sampled for recovery of Phytophthora. In the cases of one- to three-year-old seedlings from field and transplant beds, seedlings were uprooted and 10 symptomatic root tips were removed for plating.

All diseased plant tissues were surface sterilized in a 10% solution of 8.25% NaClO in sterile deionized (DI) water solution for 30 s, rinsed twice in fresh sterile DI water, and briefly dried before plating into PARPH-clarified V8 (cV8) semi-selective medium [15 g Difco agar, 33

11 ml clarified V8 juice, 10 mg Delvocid salt (50% pimaricin), 250 mg sodium ampicillin, 10 mg rifampicin sodium salt, 67 mg Terraclor (75% PCNB), 50 mg hymexazol in 1 liter water].

Cultures were allowed to incubate at 19°C in the dark for three to five days before being examined under a compound microscope to identify putative Phytophthora emanating from the tissue. Any suspected Phytophthora cultures were subcultured from the isolation plate onto fresh

PARPH-cV8 and subsequently onto un-amended cV8 medium. Cultures were microscopically examined during a seven to 10 day period for preliminary morphological classification to substantiate identity as Phytophthora prior to molecular identification. To encourage sporangia production for identification purposes, five-mm-diameter agar plugs from a given culture were flooded with non-sterile soil solution (15 g field soil dissolved in 1 liter DI water and vacuum filtered through Whatman No. 1 filter paper) and incubated at room temperature overnight under fluorescent light (Jeffers and Aldwinckle 1987). A subset of resulting sporangia were scored for shape, average size, caducity, and papilla formation. Gametangia formation and presence\absence of spores on solid media and soil solution were also noted.

DNA Extraction, polymerase chain reaction (PCR), and species identification.

To compliment morphological identification, the internal transcribed spacer (ITS) region of the nuclear rDNA and the cytochrome c oxidase I (cox I) region of the mitochondrial DNA were amplified and sequenced for molecular verification. Phytophthora cultures were prepared for DNA extraction by growing for three to five days on sterile cellophane rounds layered over fresh plates of PARPH-cV8 media. This process encourages hyphae to grow on top of the cellophane layer in order to minimize inclusion of agar medium into the reaction tubes during extraction. DNA was extracted using the QIAGEN DNeasy Plant Mini Kit (QIAGEN, Inc.,

Valencia, CA, USA) following manufacturer’s instructions. DNA and PCR products were stored

12 short-term (<1 month) at 4°C. ITS regions were amplified using ITS6F and ITS4R primers

(Cooke et al. 2000). PCR conditions for ITS amplification included initial denaturation at 94°C for 3 min followed by 35 cycles of 30 s at 94°C, 30 s at 55°C and 1 min at 72°C, with a subsequent 10 min at 72°C. The cox I region was amplified using the OomCoxI-Levup and

OomCoxI-Levlo primers (Robideau et al. 2011). PCR conditions for cox I amplification included initial denaturation at 94°C for 10 min followed by 35 cycles of 30 s at 94°C, 30 s at 56°C and 1 min at 72°C, with a subsequent 10 min at 72°C. PCR products were purified by mixing 2 µl of

ExoSAP-IT (Affymetrix, Santa Clara, CA) with 5 µl PCR product and following the manufacturer’s thermocycler protocol of 15 min at 37°C and 15 min at 80°C. After purification, diluted 3.125µM forward primer was added to the PCR products prior to sequencing by

GENEWIZ (South Plainfield, NJ).

Consensus sequences were constructed from forward and reverse sequence data using the alignment tool in the Molecular Evolutionary Genetics Analysis (MEGA) Version 6.0 program

(Center for Evolutionary Medicine and Informatics, Tempe, AZ). These sequences were submitted to search for homology among published sequences using the National Center for

Biotechnology Information (NCBI) GenBank BLASTn and Phytophthoradb.org databases

(Benson et al. 2011; Bienapfl and Balci 2014; Park et al. 2008). Database sequence results were examined against queried sequences for base pair agreement and nucleotide homology of at least

99% for species affirmation (Moralejo et al. 2009; Parke et al. 2014; Schwingle et al. 2007;

Yakabe et al. 2009). In cases where ITS was non-informative (multiple matching sequences) the cox I sequence served as a second locus for proper identification. Species identities assigned by sequence matches were verified by ensuring that morphological characteristics were consistent with published literature (Erwin and Ribeiro 1996). The MEGA 6.0 tree explorer tool was used

13 to construct a maximum likelihood phylogram using 1,000 bootstrap replicates to compare ITS consensus sequences with published sequences for verification of species identification and clarification of taxonomic standing (Figure 1). The phylogeny was constructed using a subset of isolates that included at least one isolate per Phytophthora species from each host in each state.

Koch’s Postulates. Root inoculations for completion of Koch’s Postulates were carried out to prove pathogenicity on 12 novel combinations of Phytophthora species on fir. One-year- old seedlings of four species of fir and two-year-old seedlings of two species of fir were inoculated in a greenhouse root-inoculation experiment with a varying combination of

Phytophthora spp. (Table 2). One-year-old plug seedlings (P-0 stock) of Fraser fir, Nordmann fir, and Canaan fir (A. balsamea var. phanerolepis Fern.) were obtained from Lawyer Nursery

(Olympia, WA); along with one-year-old bareroot (1-0) seedlings of grand fir. Two-year-old (P-

1 stock) Turkish fir (A. bornmuelleriana Mattf.) and Trojan fir (A. equi-trojani (Asch. & Sint. ex

Boiss) Mattf.) were started as plugs for the first year at the Kintigh Mountain Home Ranch

(Springfield, OR), and subsequently transplanted into 1.5 liter treepots (Stuewe & Sons, Tangent,

OR) to be maintained outdoors for the second year at the Washington State University Puyallup

Research and Extension Center. Phytophthora isolates were grown for 21 days on sterilized long-grain white rice in 2-mil, polypropylene, gusseted spawn bags with microporous filter patches (Fungi Perfecti, Shelton, WA). Rice was prepared by autoclaving rice grains three times over the course of three separate days in ca. 3:4 ratio of rice grain:sterile DI water. Bags were inoculated by seeding with ten seven-mm-diameter plugs taken from the edges of five-day-old pure cultures grown on cV8 medium in a sterile laminar flow hood. Rice grains for control treatments were prepared in the same way, but seeded with plugs from an un-colonized plate. All

14 bags were sealed with an impulse sealer. Spawn bags were maintained on a benchtop at room temperature for 21 days and agitated every other day to encourage uniform colonization.

Prior to inoculation, rice grains were pulverized in a kitchen-grade food processor and screened through a 2 mm (#10) soil sieve. Rice grains were incorporated into a commercially available potting mix (Gardener’s Professional Secret, Specialty Soils, Covington, WA) at a rate of 4% (0.04 g rice per g of soil). All nursery soil was washed away from the root balls of seedlings and they were subsequently planted into Ray Leach SC10 Super Cone-tainers (Stuewe

& Sons, Tangent, OR) using infested potting media. There were five replicates for each Abies x

Phytophthora combination and each control treatment. Seedlings were maintained at 21°C and exposed to a 12 h photoperiod under 400-watt high-pressure sodium greenhouse lights for eight weeks. Light levels were measured using the Quantum meter on a LiCor LI-1600 Steady State

Porometer (LiCor, Lincoln, NE) and averaged about 178 µmol/s -1/m-2 at full intensity.

Immediately following inoculation, seedlings were subject to two 45 min drenches within a 24 h period via overhead irrigation at a rate of 0.25 ml/min to promote the saturated conditions that are conducive to PRR. Subsequent irrigation was administered twice daily for 10 min periods, once in the morning and once in the evening, for the duration of the experiment.

At the end of the incubation period, seedlings were removed from the cones and the infested potting media was washed from the roots. The extent of root rot was rated by teasing apart the roots and visually estimating the proportion of symptomatic root tips on a scale of 0 to 5, where

0 = < 10% necrotic root tips, 1 = 10% to 25%, 2 = 25% to 50%, 3 = 50% to 75%, 4 = 75% to

90%, and 5 = > 90% necrotic roots (Table 2). Ten symptomatic root tips from each tree were plated onto PARPH-cV8 selective medium to confirm recovery of the pathogen. All resulting

15

Phytophthora cultures were subcultured to un-amended cV8 media upon re-isolation from symptomatic roots, and were matched to the original isolate by morphology and ITS sequence.

Results

Survey Sites. Of the 32 Christmas tree plantations visited in the PNW, western, northeastern, and southeastern regions of the U.S., Phytophthora was recovered from a total of

36 fir trees in 24 sites, yielding 105 total isolates (Table 1). Michigan cultures were isolated from the soil surrounding seven host trees in five counties and Wisconsin cultures were received from five host trees in three counties, comprising 12 total Phytophthora isolates from the Great Lakes

Region (Table 1). The maximum likelihood phylogram that was constructed to infer confidence in species identifications verified that collected isolate identifications were statistically similar to known reference sequences and provided information about the distribution of Phytophthora clade groups in U.S. Christmas tree farms (Figure 1).

P. cambivora was the most commonly isolated species in the PNW region represented by western Washington, western Oregon, and northern Idaho. Nearly 90% of the sampled trees in

Oregon and Washington were mature (7- to 10-year-old) noble fir; on which, disease was easily identified by flagging lower branches and large bole cankers. The majority of the samples from noble fir were isolated from the lesion margins between healthy and necrotic tissues on the lower stems and crowns. A single P. megasperma isolate was recovered from the roots of a two-year- old transplanted noble fir in a Washington site that is commonly flooded from a nearby retention pond that previous sampling has indicated harbors P. megasperma. Also in Washington, diseased

Fraser fir seedlings in a nursery transplant bed yielded putative P. pseudosyringae Jung &

Delatour., but Koch’s Postulates have not yet been completed to verify this interaction. A grand fir sample from southwestern Oregon was found to have aerial infections caused by P.

16 gonapodyides on lower branches and twigs, comprising the only species aside from P. cambivora recovered from Oregon. Host species sampled in Idaho included concolor (white) fir and grand fir, but collections yielded only a single isolate of P. megasperma from the roots of a diseased grand fir in a stand of mature dead and dying trees near the base of a slope at one farm.

In the western region, P. cinnamomi was the only Phytophthora isolated from root tissues of a variety of hosts at several northern California farms. The hosts sampled in California included a diversity of age classes and species including the exotic Trojan and Nordmann firs and native concolor fir. Other symptomatic trees sampled in California included Shasta red fir and Turkish fir, but sampling from these individuals did not yield Phytophthora.

In the northeastern region, P. taxon kelmania was the most frequently isolated species from both Connecticut and New York. The second most-commonly isolated species was P. pini, followed by P. plurivora and P. sansomeana. P. cactorum was isolated only once from a single symptomatic Fraser fir seedling in a Connecticut transplant bed. The predominant fir species sampled in the northeastern region was Fraser fir, with trees of all age classes proving to be viable hosts for Phytophthora.

P. taxon kelmania was also the most commonly isolated Phytophthora in North Carolina, followed closely by the historically predominant P. cinnamomi. These two species were found on Fraser fir, but were also commonly isolated from Turkish and Trojan firs planted at multiple sites in northern North Carolina. Additional Phytophthora species isolated from Fraser fir in

North Carolina include P. pini and P. cryptogea.

Cultures from collections in Michigan and Wisconsin included isolates of species that were not observed in other regions, including P. europeae Hans. & Jung and P. nicotianae Breda de Haan.

17

Koch’s Postulates. The results of these trials provided proof of pathogenicity for many new Phytophthora-Abies combinations (Table 2). Foliar symptoms observed on inoculated seedlings included wilt and/or necrosis of newly-expanding shoots, a failure to break bud, and overall seedling mortality. Visual root rot ratings ranged from a high of 4.8 for P. pini on Fraser fir to a low of 2.0 (Table 2). Commonly, some darkened roots were observed on the non- inoculated control seedlings, resulting occasionally in ratings that registered as high as a 2.0; however, no foliar symptoms were observed on control seedlings and no Phytophthora were isolated from any darkened root tips of non-inoculated seedlings. Phytophthora were re-isolated from symptomatic roots on 5/5 inoculated seedlings for each host-pathogen challenge in both trials. All re-isolated cultures from inoculated seedlings were consistent in morphology and ITS sequence to the isolates that were originally used for inoculation, confirming pathogenicity of the

Phytophthora species on their corresponding hosts (Table 2).

Discussion

Assessments of the diversity in the genus Phytophthora estimate that there are several hundred species, with a large percentage associated with diseases of forest and nursery trees

(Brasier 2009). The survey reported here provides insight into the distribution and community structures of the Phytophthora species that are affecting Abies in U.S. Christmas tree farms.

Regional variation in the occurrence of a particular species of Phytophthora may depend on annual rainfall, median temperatures, and the host species present, but local Phytophthora communities may also be a function of the nurseries that produce the seedlings. It has become common for seed to be contracted to nurseries in distant regions to be grown as containerized seedlings and then distributed back to farms to be transplanted into outdoor beds, resulting in involvement from a greater number of nurseries and packing facilities. The nationwide

18 circulation of conifer nursery stock could be a potential avenue for cultivation and dissemination of root-rotting Phytophthora and may have the capacity to increase or dictate the diversity of

Phytophthoras present in a particular region. Nursery location may limit the presence of a particular Phytophthora; for example, species that are favored by warm soils may not thrive in nurseries in cooler climates. Thus, the recipient states of seedlings grown in those nurseries may not observe those Phytophthora spp. as often in their plantings. Poor survival of nursery-borne

Phytophthora has also been demonstrated when infested seedlings are outplanted from nurseries into locations with uncomplimentary environments (Hansen et al. 1980; Roth and Kuhlman

1966). Conversely, the expansion of plant trade and movement may increase local Phytophthora diversity by introducing species into novel areas conducive to their survival and proliferation. In the current survey, there was a greater diversity of Phytophthora affecting fir in the eastern and central production areas. This may reflect environmental parameters such as large seasonal temperature fluctuations and ample rainfall during the growing season in contrast to the year- round temperate conditions and dry summers experienced in the western regions, but also may be a result of an increased number of nurseries producing Fraser fir for planting in eastern farms.

In western Washington and Oregon, noble fir was the most commonly sampled Abies species. The observed dominance of P. cambivora on noble fir provided an updated representation of the Phytophthora community on this host. P. cambivora is a pathogen of temperate host species including a variety of deciduous hardwood and fruit trees in the northern latitudes of Europe and the contiguous U.S., and is infrequently reported from warmer, drier regions (Day 1938; Erwin and Ribeiro 1996; Mircetich and Matheron 1976; Talgø et al. 2006,

Vannini and Vettraino 2001). This species does poorly at temperatures exceeding 30°C and is therefore well adapted to the cool, wet conditions in the PNW (Erwin and Ribeiro 1996).

19

Chastagner et al.’s 1990 survey of noble fir in 30 western Washington and Oregon Christmas tree farms indicated a community of at least seven different species of Phytophthora, dominated by P. cambivora, P. megasperma, P. cryptogea, and P. gonapodyides (Chastagner et al. 1995).

Although the geographic coverage in the present study was not as comprehensive as

Chastagner’s survey, one may speculate that the current lack in diversity of Phytophthora on noble fir, save P. cambivora, could reflect changes in the conifer nurseries that grow and provide noble fir stock to growers in the Pacific Northwest. Historically, a number of U.S. Forest Service nurseries supplied bare-root noble fir seedlings to growers in Oregon and Washington. Many of these nurseries were established on bottomland river sites prone to flooding events and the saturated soils that are conducive to supporting diverse Phytophthora populations. As the number of these nurseries has decreased, the remaining facilities producing Abies seedlings are generally located on upland and interior sites. Changes in nursery location, as well as improvements in nursery management practices and a shift in production from bare-root to container-grown Abies seedlings may have contributed to the observed reduction in

Phytophthora spp. diversity on noble fir in the last 20 years. In spite of these observations, participating growers in this survey were queried about the origins of their seedlings, but in the breadth of this study, no distinct patterns were evident relating to the sources of planting stock and Phytophthora species present.

When PRR was observed on other host species in Washington and Oregon, such as grand and Fraser firs, the diversity of Phytophthora spp. obtained from this region was augmented with the addition of P. gonapodyides and P. pseudosyringae. P. gonapodyides is a constituent of riparian and is commonly associated with woody hosts (Blair et al. 2008; Brasier et al. 1993; Kroon et al. 2012). This species, along with P. megasperma, have been isolated from

20 aqueous environments including stream water and retention ponds (Brasier et al. 1993). The aerial P. gonapodyides twig infections on grand fir in Oregon and the P. megasperma infection on noble fir near a retention pond in Washington may have resulted from irrigation using contaminated water. P. pseudosyringae has been described infecting oak species in Europe, but is also weakly pathogenic on California bay laurel (Umbellularia californica) (Hook. & Arn.)

Nutt, tanoak (Notholithocarpus densiflorus) (Hook. & Arn.) Manos, Cann., & Oh, and coast live oak (Quercus agrifolia) Nee in the mixed-evergreen of northern California and southern

Oregon (Jung et al. 2003; Linzer et al. 2006; Murphy and Rizzo 2006; Reeser et al. 2009;

Wickland and Rizzo 2006). The detection of this species in a western Washington transplant bed on Fraser fir seedlings may have been the result of an introduction on planting stock since these seedlings were planted into fumigated ground. This occurrence may comprise a new report of this species on fir, but Koch’s Postulates have not yet been performed to confirm pathogenicity.

Preceding this survey, little information was available relating to PRR communities affecting fir in Idaho, and current sampling yielded few isolates. In this production region, there is a large market for PRR-tolerant Pinus and Picea Christmas trees in addition to a small selection of Abies comprised mostly of white and grand firs. Since few of the more highly PRR- susceptible species such as noble and Fraser firs are grown in Idaho farms, there is less disease, and it is harder to spot visually. Environmental parameters in this region may also be unfavorable for disease development given extreme summer dryness, general lack of irrigation, and cold winter conditions. This may result in the relegation of PRR outbreaks to only certain years, or times of year when the environmental conditions are most conducive to disease development.

Broadening the geographic area and seasonal time frame of sampling may provide a more complete picture of Phytophthora communities in Idaho.

21

The presence of P. cinnamomi on fir in California and North Carolina was expected, as these regions have historically dealt with this pathogen (Benson and Grand 2000; Frampton and

Benson 2004, 2012; Garbelotto et al. 2006; Grand and Lapp 1974; Zentmyer 1977). Although P. cinnamomi occurs on a variety of hosts over a wide climatic range, it is well adapted to warm conditions and is a pathogen of subtropical plants including avocado (Persea Americana) Mill., pineapple (Ananas comosus) (L.) Merr., and kiwi fruit (Actinidia chinensis) Planch. in southern

California, jarrah trees ( marginata) Donn. ex Sm. in Western Australia, and in the southeastern US and New Zealand (Campbell 1948; Erwin and Ribeiro 1996; Mehrlich 1936;

Newhook and Podger 1972; Wager 1942; Zentmyer 1977). In both California and North

Carolina, warm soil conditions during much of the year provide temperatures conducive to the survival and proliferation of P. cinnamomi. Other features of these growing regions that encourage P. cinnamomi infection include ample moisture that coincides with warm soils, in the form of natural rainfall in the southeastern U.S., or as overhead irrigation in growing operations in northern California. In contrast to the historical predominance of P. cinnamomi causing PRR in North Carolina, the current survey detected P. taxon kelmania more commonly in this state than P. cinnamomi, and also found it to be affecting PRR-tolerant Turkish fir in addition to the commonly-affected Fraser fir host. P. taxon kelmania is a member of the Phytophthora clade 8a, along with P. drechsleri and P. cryptogea, and shares many of the same characteristics of these two including heterothallism, non-papillate sporangia, and morphological resemblance (Blair et al. 2008; Kroon et al. 2012; Moralejo et al. 2009; Mostowfizadeh-Ghalamfarsa et al. 2010).

Although P. drechsleri and P. cryptogea have long been recognized as common PRR species, P. taxon kelmania has only been acknowledged by the scientific community since the early 2000’s and is currently being proposed as a novel candidate species. (Moralejo et al. 2009). While more

22 intensive sampling of farms in North Carolina would help to substantiate which Phytophthora species are currently the most pervasive in this region, the emergence of P. taxon kelmania, as well as the detection of others such as P. cryptogea and P. pini, suggest an increased diversity of

PRR-associated Phytophthora in contrast to a landscape historically dominated by P. cinnamomi.

P. taxon kelmania was also a common member of the PRR communities in the northeastern states of Connecticut and New York, and was received in the collection from Wisconsin, suggesting a geographical derivation from eastern areas. Collections from Abies in the eastern regions indicated more diverse Phytophthora communities in comparison to the western U.S., despite being sampled from a relatively narrow host range dominated by Fraser fir. This diversity may again be attributable to a Phytophthora-conducive growing season including summer rainfall, but also to the nationwide circulation of planting stock from an increasing number of nurseries producing Fraser fir seedlings in response to an increased demand for this host species.

The information from this survey has the potential to benefit the growers and purveyors of the conifer seedling and Christmas tree industries by providing valuable information about contemporary host and pathogen ranges, including first reports about new findings. Growers cultivate host species in response to market demands, but have also been trying to plant more

PRR-tolerant Abies spp. as a cultural method for combatting Phytophthora issues. An ideal solution for avoidance of seedling-borne Phytophthora would be to purchase certified disease- free seedlings for planting; however, the delay between root infestation and the onset of visible foliar symptoms often makes it difficult to ensure that nursery-grown material is free of contamination. Therefore, up-to-date knowledge of the community structure of root-rotting

Phytophthora in local areas will assist growers in assessing the risk associated with certain host species substitutions on their farms. Validation of novel host-pathogen combinations and

23 subsequent dissemination of first reports help researchers provide refined expertise to growers and bridge the gap between technical research and public knowledge. The data presented in this paper corroborate an underlying concern over a perceived increase in the diversity of

Phytophthora in certain regions and inspire questions into the factors that may be contributing to these changes. It is suggested that a current survey of PRR in U.S. conifer nurseries may aid in pinpointing sources of contamination and elucidating channels of Phytophthora distribution on

Abies.

24

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Talgø, V., Herrero, M., Toppe, B., Klemsdal, S., Stensvand, A. 2006. First Report of root rot and

stem canker caused by Phytophthora cambivora on noble fir (Abies procera) for bough

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Chapter 2. Interactions Between Root Rotting Phytophthora, Abies, and Environment

Increased demand by Christmas tree consumers for Phytophthora Root Rot (PRR)- susceptible fir species and the necessity for growers of live Christmas trees to remain competitive with artificial tree industries has spurred interest in approaches to reclaim

Phytophthora-infested acreage. Active research areas include the potential development of molecular tools to identify PRR resistance within Abies populations and the characterization of host phenotypes to improve recommendations for host species substitutions as a means of disease avoidance. The ability to definitively assign a resistance phenotype to a given host is fraught with complicating factors. One concern is that PRR is caused by multiple species of

Phytophthora that vary in aggressiveness, climatic adaptations, and geographic distribution.

Consequently, the characteristics of local Phytophthora communities may differ widely from region to region (McKeever and Chastagner 2016). Additionally, the many species of fir that are utilized for Christmas trees differ in their inherent ability to resist infection. The mechanism of resistance, whether chemical or physical, is currently unknown; however, it has been observed that exotic Abies species from Eastern Europe and Asia are more resistant to disease than native

North American Abies species (Frampton 2005; Frampton et al. 2012). Furthermore, abiotic environment may influence symptom expression in the host and performance of the pathogen; for example, factors such as temperature and soil moisture contribute to host transpiration rates and root health, and also influence proliferation of the pathogen and spore dissemination through the soil. For these reasons, research performed to evaluate host performance in different geographic areas can result in conflicting information regarding the ability of a given host to resist disease under local conditions (McKeever and Chastagner 2016).

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Few experiments have been designed in such a way as to address variability across the broad geographical areas in which fir are grown and PRR is a problem. Researchers at North

Carolina State University have extensively investigated the dynamics of various Abies species that are afflicted with disease caused by Phytophthora cinnamomi Rands, which is by far the most pervasive Phytophthora species in growing regions in the southeastern U.S. (Benson and

Grand 2000; Grand and Lapp 1974). Findings indicate little-to-no resistance to P. cinnamomi in

Fraser fir (A. fraseri), which is the most highly demanded and widely grown Abies species in the eastern and central U.S. (Frampton and Benson 2004; Hinesley et al. 2000; Shew and Benson

1981). Testing of 32 native and exotic Abies species for resistance to P. cinnamomi indicated good survival of momi (A. firma Siebold & Zucc.) and Himalayan (A. pindrow (Royle ex D.

Don) Royle) firs, and less than 70% losses in Siberian fir (A. siberica Ledeb.) and Turkish fir (A. bornmuelleriana Mattf.); however, the remaining 28 Abies species displayed little resistance to

P. cinnamomi (Frampton and Benson 2012). Similar studies have corroborated these findings when challenging Abies species with P. cinnamomi (Benson et al. 1997; Frampton et al. 2012;

Hinesley et al. 2000), but there have been few complementary studies done to test these findings with other Phytopthora species under different environmental conditions. Chastagner and others at Washington State University performed a study to evaluate 11 Abies species for resistance to seven Phytophthora spp., finding clear evidence that the Phytophthora species used varied considerably in their ability to cause disease (Chastagner et al. 1990). Campbell and Hamm

(1989) found similar variation in Phytophthora spp. aggressiveness when inoculating 10 non-

Abies Pacific Northwestern conifers with five common PRR-causing species. Hamm and Hansen

(1982) tested multiple isolates of nine Phytophthora species isolated from the Pacific Northwest on a set of Abies and non-Abies conifer seedlings, and found considerable variation in disease

33 ratings among the various Phytophthora species and even among different isolates of the same

Phytophthora species. These research results have made it clear that true fir vary in their relative sensitivities to PRR and that a great deal of variation exists among the species of Phytophthora that cause disease.

A plausible management technique would be to tailor host species substitutions to local environmental conditions and pathogen populations as a means of minimizing losses; however, variation in host symptom expression in response to different temperatures, moisture patterns, and species of Phytophthora is poorly understood. The development of a genetic marker system and subsequent detection of resistant individuals could spur the initiation of Abies breeding programs to provide disease-resistant planting stock for growers; however, such a system will need to be applicable to multiple fir species over a broad geographical scope. By cultivating an understanding for the intricacies of the system, researchers can work toward developing improved management techniques and alternative methods for avoiding PRR in all afflicted regions. To satisfy these goals, a multifactorial study incorporating combinations of the interacting factors in the PRR pathosystem was implemented to understand how modification of various parameters may affect disease development, symptom expression, and pathogen virulence.

Materials and Methods.

Experimental Design. Four species of Phytophthora were used to challenge 7 Abies spp. at two temperature ranges. A cool temperature range of 15 - 21°C (further referred to as 15°C) was chosen to simulate environmental conditions that may be encountered year-round in more temperate geographic locations such as the Pacific Northwest, or at cooler times of the growing season in other locations. A warm temperature range of 27 - 32°C (further referred to as 27°C)

34 was chosen to suggest the typical summertime temperatures encountered in many U.S. growing regions. Assessing PRR at two different temperatures required that the experiment be arranged in two separate greenhouses. Since variation exists among isolates within a given Phytophthora species (Eggers et al. 2012; Granke et al. 2011), the experiment was partitioned into three simultaneously-running experiments, each employing a different isolate of the four Phytophthora species. Five replicate blocks of each Phytophthora-Abies combination were executed at each temperature.

Each of the four species of Phytophthora plus one non-inoculated control treatment were randomly assigned within each replicate block. Within each Phytophthora species subdivision, three geographically distinct isolates of that given Phytophthora were randomly distributed.

Inoculum of each species × isolate treatment (or a non-inoculated treatment) was applied to a set of seven different fir species. Each species of fir was made up of five individual seedlings representing an experimental unit; of which, collected data points were averaged to generate a single measurement.

Seedlings. The seven species of Abies assessed in this study represent the most commonly-grown and economically important Christmas tree species for U.S. markets. Five of the species, Fraser, noble (A. procera), white (A. concolor (Gordon) Lindley ex Hildebrand),

Canaan (A. balsamea var. phanerolepis Fern.), and balsam firs (A. balsamea (L.) Mill.), are native to North America. The two remaining species used in this experiment are native the

Caucasus Mountains of Northern Turkey and Western Eurasia and belong to the closely related

Abies nordmanniana complex. Seedlings were obtained in February 2014 and 2015 from the

Weyerhaeuser Corporation’s Rochester Nursery (Rochester, WA). The five North American species were grown as one-year old containerized stock (P-0). Nordmann and Turkish fir were

35 obtained as two-year old bareroot seedlings (P+1). All seedlings were received frozen and physiologically dormant. In June of each year, thawed seedlings were transplanted into 262 ml

D16L Deepots (Stuewe & Sons, Tangent, OR) using a custom conifer potting mix prepared by

Specialty Soils, Inc. of Covington, WA (Table 1). Planted seedlings were placed into D50T 50- cell capacity support trays and maintained outdoors from June through October on elevated -and-cinder benches underneath 50% shadecloth. Irrigation was initiated via overhead sprinkler two times daily for 20 minute increments for the first two weeks during transplant establishment and subsequently thereafter during hot spells (> 29ºC). A regular outdoor watering schedule was maintained once-daily with a 15 minute duration during temperate weather. Two times during the outdoor storage period, seedlings were treated with fludioxonil (Medallion®,

Syngenta Crop Protection, Inc., Greensboro, NC) applied as a spray until runoff at a rate of 56.7 g (2 oz) per 378.5 liters (100 gal) water in order to prevent Botrytis development during storage.

In early October, as seedlings were entering dormancy following terminal bud development, the seedlings - still potted in the Deepots - were packaged into wax-coated seedling boxes to undergo an artificial vernalization period at 3°C in the dark for 800 hours (ca. four weeks). This vernalization period allowed for experiments to be conducted on dormant seedlings and ensured consistent budbreak upon resumption of physiological activity. Upon termination of the vernalization period, seedlings were removed from the boxes, labeled with pull-through tree tags, and randomized into two greenhouses where experiments were conducted. During the three- week period between introduction into the greenhouses and initiation of the experiment, greenhouse conditions were kept cool at 12 to 15°C with no artificial lighting and drawn shadecloth in order to allow for easy transition from cool storage temperatures to the warm,

36 lighted temperatures that would be implemented during the experiment. Irrigation during this transitional period was applied once-daily for 10 min durations at a rate of 0.25 ml/min.

Selection of Phytophthora Species & Isolates. Phytophthora cultures used for inoculum in this study were sourced from a nationwide collection of root-rotting Phytophthora from Abies in Christmas tree farms that was conducted during a two year period from 2012-2013 (McKeever and Chastagner 2016). The four most-commonly occurring Phytophthora species from this survey were selected to comprise the inoculum for this greenhouse study. Since several isolates of each of the four Phytophthora species were collected, virulence testing was performed to acquire the three most aggressive isolates of each species to test concurrently in the planned greenhouse experiment. Table 2 provides information on the origins and NCBI identities of each isolate employed in the virulence assessment. Seedling stem inoculations were chosen to measure virulence, as this is an established method for Phytophthora that correlates well to aggressiveness on roots (Bhat et al. 2006; Weiland et al. 2010).

Prior to assessing virulence, all collected isolates of the four Phytophthora spp. of interest were inoculated onto noble fir branches in a preemptive effort to maximize wildtype virulence after repeated subculturing on artificial media (Hodgson and Sharma 1967). For restorative inoculations, all isolates of a single Phytophthora species were inoculated onto different branches of the same field-grown noble fir. Inoculations were made in the 2nd year’s internodal region of the branch by scoring the with a five mm-diameter cork borer and peeling back the thin bark layer to expose the phloem underneath. A five mm-diameter plug from an actively growing five day-old culture on V8 media was applied facedown to the phloem and the bark layer was replaced. Inoculation sites were sealed with sterile petroleum jelly, wrapped in moist sterile cotton, and fastened with Parafilm (Bemis, Neenah, WI). Inoculation sites were allowed to

37 incubate on the tree for 14 days at ambient outdoor temperatures averaging 19°C (diurnal fluctuations ± 7°C) with 71% average relative humidity. After 14 days, samples were taken from the margins of the lesioned and healthy tissues surrounding the inoculation sites, surface sterilized with a 10% solution of 8.25% NaClO in sterile deionized (DI) water, and plated into

PARPH-clarified V8 (cV8) semi-selective medium [15 g Difco agar, 33 ml clarified V8 juice, 10 mg Delvocid salt (50% pimaricin), 250 mg sodium ampicillin, 10 mg rifampicin sodium salt, 67 mg Terraclor (75% PCNB), 50 mg hymexazol in 1 liter water]. Resulting cultures were transferred to unamended cV8 medium.

Virulence testing was performed on actively growing two-year-old bareroot noble fir seedlings that had been transplanted into one liter MT38 Treepots (Stuewe & Sons, Tangent,

OR) and maintained outdoors for four weeks prior to use. Virulence testing was performed in

August 2014 in a randomized complete block design with three seedlings constituting an experimental unit and three replications (blocks) of each treatment. The experiment was repeated once. Height and caliper measurements were recorded for each seedling in an effort to choose uniform stem diameters for inoculations. Inoculations were performed on the stem of the seedling approximately 12 cm above the soil line and techniques were identical to the restorative isolations described above. Treatments were randomized within each block and incubated in a

17°C greenhouse for seven days with shadecloth drawn and no supplementary lighting.

Overhead irrigation was applied for 10 minutes, once a day, at a rate of 0.25 ml/min. After seven days, resulting lesions were exposed by carefully scraping back the bark starting at the point of inoculation and extending to the perimeters of the lesion. Digital caliper measurements of lesion length were recorded.

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Of the final 12 selected isolates (three isolates of each of the four species of

Phytophthora), radial growth measurements were assessed at two different temperatures in anticipation of the implementation of two temperatures for the greenhouse study. The greenhouse experiment was to be conducted at both a cool temperature range of 15 to 21°C and a warm temperature range of 27 to 32°C in two separate greenhouses; thus, radial growth of the

Phytophthora isolates at these temperatures was assessed to document differences. Plugs (four mm-diameter) of each isolate were grown on cV8 medium in three replicate Petri dishes in incubators set to 17°C and 29°C. Radial growth (mm) in two opposing directions was recorded

24 hours after plating and then daily for up to eight days. Measurements for opposing directions of each plate and for the three replicate plates were averaged for each isolate. This assay was repeated once.

Inoculation. Colonized rice grains were used to apply experimental treatments to seedlings (Holmes and Benson 1994). For each treatment, eight grams of long grain white rice was mixed with 5.76 ml deionized (DI) water in a 250 ml flask, autoclaved twice, and seeded with six three-mm-diameter plugs taken from the edges of four-day-old cultures grown on cV8 medium. Non-inoculated treatments received six plugs of non-colonized cV8. Flasks were incubated at room temperature for 21 days, with agitation on alternating days to break up rice mats and evenly distribute inoculum. A separate set of 15 flasks (three isolates each of four

Phytophthora spp. and one non-colonized control) was produced for each replicate block totaling

75 flasks for each greenhouse. Seedlings were inoculated by inserting a single rice grain in each of three holes in the planting substrate that were positioned around the stem of the seedling and angled toward the rootball, to a depth of 10 cm. A single flask of inoculum was used to inoculate all seven fir spp. receiving a given treatment in a single block. After eight weeks of incubation, a

39 second inoculation was performed on living trees following the above protocol. This second inoculation was implemented as a precaution to ensure that surviving seedlings were not simply disease escapes resulting from a poor first inoculation.

Incubation Conditions. For the first five days immediately following both inoculation periods, overhead irrigation was applied at a rate of 0.25 ml/min for 60 min durations, twice per day, to create saturated soil conditions that are conducive to PRR (Kuan & Erwin 1980; Rhoades et al. 2003). After this initial saturation period, irrigation was reduced to a 20 minute period, once daily. To comply with USDA APHIS regulations, irrigation runoff was collected and disinfested with 8.25% NaClO. A 12 h photoperiod was maintained throughout the duration of the experiment using 400-watt high-pressure sodium greenhouse lights at an average intensity of

178 µmol/s -1/m-2. Greenhouse temperatures were programmed to remain within dictated degree ranges throughout a 24 hour period with no predetermined diurnal fluctuations. Cooling was achieved through automatic activation of exhaust fans and evaporative cooling. To preempt the possibility of infestation by Botrytis spp., trees were treated once during shoot elongation with fludioxonil at a rate of 56.7 g (2 oz) per 378.5 liters (100 gal) and air circulation was maintained using four horizontal airflow fans positioned overhead in the four corners of each greenhouse.

Ambient temperature and relative humidity were recorded with HOBO Pro data loggers (Onset

Computer Corp., Bourne, MA) at the western and eastern ends of each greenhouse. Soil temperature was measured using HOBO 4-channel analog data loggers with probes inserted 7.6 cm into the soil of two individual seedlings in each block. Seedlings were undisturbed for eight weeks following the first inoculation and then again for an additional 8 weeks following the second inoculation for a total duration of 16 weeks for the experiment. This experiment was repeated once.

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Assessments. The onset of budbreak for each species was noted, and foliar assessments were made biweekly beginning at the onset of symptoms in the most susceptible species (ca. 19 days post-inoculation) to estimate PRR incidence. Foliar ratings were made on an ordinal scale of 1 = alive (green foliage, fully expanded new growth, full turgor), 2 = failing (wilt, chlorosis, failure to break bud, desiccation, partial necrosis), and 3 = dead (fully necrotic foliage or severely wilted past the point of likely recovery).

At the 16 week conclusion of the experiment, seedlings were destructively processed to assess disease severity on the roots. All seedlings were carefully removed from Deepots and root systems were washed free of potting media, taking care to retain as much root tissue as possible in severely damaged seedlings. The percent of rot on the root system was visually assessed on a

1 - 4 scale where 1 = < 25% rot, 2 = 25% - 50% rot, 3 = 50% - 75% rot, and 4 = > 75% rot. To estimate relative reductions in comparison to non-inoculated treatments, root systems were detached from the stem at the collar of the highest root and dried at 70°C for 72 hours prior to weighing. To verify Phytophthora as the causal agent of mortality, root tip isolations were made on a subset of seedlings from each treatment in each block.

Data Analysis. Analyses of variance of the various data sets generated by this research and mean separations among treatments were performed with SAS 9.4 (The SAS Institute, Cary,

NC). Graphical representations of data were achieved using R Statistical Software (The R

Foundation, Vienna, Austria). Analyses of variance of lesion size data from virulence testing assays were performed to compare isolates within each of the 4 Phytophthora species individually, using PROC MIXED procedures with post-hoc mean separations using Tukey’s

HSD. Data from the two trials were combined. For the greenhouse screening, data from the two temperature greenhouses were assessed separately because of the lack of replication of

41 temperature within a single year’s trial. The three isolates of each Phytophthora species were also analyzed separately in each analysis, due to the selected method of physical randomization and spatial setup within each greenhouse. For all comparisons of response variables among hosts and treatments, data from the five-tree experimental unit for each fir species in each treatment were averaged to constitute a single measurement.

Biweekly foliar assessments from the greenhouse screening were summarized as a time course of percent mortality within each host-treatment combination and used to construct disease progress curves (Madden et al. 2007). Mortality over time was used to calculate area under the disease progress curve (AUDPC), which in turn was used to compare mortality among host species in each treatment. AUDPC measurements were log transformed to achieve normality within a host species and linearize the data. The root rot severity categorical classes (1 - 4) were converted to their corresponding percentage ranges and the midpoint value of each range was utilized for parametric statistical analysis (Campbell and Neher 1994; Horsfall & Barratt 1945).

Dry weights, in grams, of the dried root systems were expressed as percentages of the control

(non-inoculated) treatments. ANOVA tests were performed to assess differences in each response variable among the different fir species within each treatment, as well as among the different treatments for each fir species individually. Pearson’s product-moment correlation

(PPMC) was used to assess harmony among the three response variables from the greenhouse

Abies screening. For PPMC analysis, data from all trials, treatments, and temperatures were combined to understand how well the response variables represented each other; but were also examined by each host species individually to assess differences. Root rot rating and dry weight response variables were regressed against AUDPC to gauge the predictive power of using data from these response variables to forecast disease progression.

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Differences in radial growth rate of Phytophthora cultures at 15°C and 27°C were compared using paired two-sample t-tests with data from both trials combined. Unequal variances were assumed due to differences between the two temperatures in the number of days required for a given culture to reach the perimeters of the Petri plate.

Results

Selection of Phytophthora Inocula & Virulence Testing. The four most-commonly occurring species of Phytophthora from the nationwide collection included P. cambivora (Petri)

Buisman (11 isolates), P. cinnamomi (seven isolates), P. kelmania (seven isolates), and P. pini

Leonian, 1925 Emend. Gallegly, Hong, Richardson & Hong 2008 (four isolates) (McKeever and

Chastagner 2016). Three isolates were selected from each species based on resulting lesion sizes on noble fir stems and geographic distinctiveness (Figure 1). Isolate numbers 2, 4, and 5 were selected for P. cinnamomi, isolates 1, 5, and 8 were selected for P. cambivora, isolates 1, 4, and

6 were selected for P. kelmania, and isolates 1, 2, and 4 were selected for P. pini (Figure 1).

Correlations of the Response Variables. PPMC analysis indicated strong correlations among the three assessments, coercing rejection of the null presumption. Root rot ratings, expressed as the midpoint value of the percentage range, were very strongly positively correlated with AUDPC (r=0.93, N=182, p<0.0001) indicating that a higher percentage of root rot on the root ball was strongly related to more severe and rapid disease progress. The dry weights, expressed as a percentage of the control seedling dry weights, were also linearly correlated with

AUDPC, but in a negative direction (r= -0.71, N=182, p<0.0001), indicating that greater disease was associated with a smaller amount of root biomass. When evaluating the relationship between the amount of rot on the rootball and the dry weight biomass, a strong negative

43 relationship was again observed (r= -0.79, N=182, p<0.0001) suggesting that greater disease severity results in lower dry weights, presumably due to decimation of the plant tissue.

Because of intrinsic variation among the different species of Abies in root morphology, size, and the ability to withstand disease and/or regenerate roots, the correlation of these three response variables was also analyzed by host species in order to assess whether the overall correlations held up, or if the perceived relationships were spurious. In general, the relationships analyzed on an individual host basis tended to be just as strong as the overall assessments of correlation, aside from slightly weaker, though still significant, correlations between dry weights

(% of control) and AUDPC in white fir and noble fir (Table 3).

Multiple Regression of the Response Variables. Regression modeling to test the null assumption that root rot severity and biomass would be inadequate to understand variability in

AUDPC was significant (p<0.0001); however, an insignificant p-value for the dry weight variable indicated that only the root rot rating could be relied upon to sufficiently predict future values of AUDPC.

To further examine this causality and to assess the usefulness of the dry weight response variable on an individual-host basis, this same model was applied to the data collected for each

Abies species individually (Table 3 and Figures 2 & 3). Significance of the overall models for each individual test indicated suitability of the model; however, dry weight again proved to be an insignificant predictor of AUDPC for noble fir, Nordmann fir, and Turkish fir (Table 3).

Comparing Dry Weight Biomass Among Host and Phytophthora Species. Because of intrinsic differences in the sizes of the root systems between different species of Abies, dry weights in grams were expressed as percentages of the control seedling weights in order to standardize observations and allow comparisons between host species. The scarcity of statistical

44 significance among hosts limited the ability to specify trends based on dry weight biomass. Mean separations (Figure 4) indicate that any detectable significant differences were generally driven by weight reductions in noble and Fraser firs. Canaan and balsam firs suffered notable reductions in root biomass when challenged with P. kelmania and P. cinnamomi at warm temperatures, but were largely unaffected by any treatment in the cool greenhouse. White fir biomass measurements tended to cluster with the susceptible noble and Fraser firs in the P. kelmania treatment, but were more comparable to resistant species such as Nordmann, Turkish, and

Canaan when challenged with other Phytophthora species. In general, reductions in root biomass were evident among all hosts in P. cinnamomi isolates 2 and 3 treatments and in all treatments at warmer ambient temperatures.

When comparing dry weight biomass among Phytophthora spp. by host (Figure 5), the data suggest that P. cinnamomi and P. kelmania are responsible for more severe weight reductions than P. cambivora or P. pini. Phytophthora cambivora was generally weak on most species aside from noble fir. Phytophthora pini was moderately virulent, generally clustering statistically with P. cambivora, but resulting in slightly lower biomass measurements on most hosts. The inherent susceptibility of noble fir was evidenced by reductions in biomass that differed significantly from the control percentage (100%) for nearly every Phytophthora treatment. For the remaining five hosts, only P. cinnamomi and P. kelmania reduced root biomass to a level that was significantly different from non-inoculated treatments.

Comparing Root Rot Rating Among Host and Phytophthora Species. In general, noble and Fraser firs suffered the greatest root damage and Nordmann and Turkish firs suffered the least (Figure 6). Canaan fir tended to cluster statistically with the less susceptible species, rarely showing difference from Nordmann and Turkish firs (Figure 7). Observationally, balsam

45 fir appeared to suffer somewhat less damage than white fir, although comparison of the means indicated that the differences were rarely significant (Figure 7). Balsam was generally more intermediate than white fir, commonly clustering statistically with both the susceptible and resistant species. White fir, although occasionally displaying commonality between both susceptible and resistant species, tended to suffer root damage that was more reflective of the performance of noble and Fraser firs (Figure 7).

Observational comparison of root rot severity due to Phytophthora treatment for each host (Figure 6) suggested a trend of greater damage on host roots inflicted by P. cinnamomi, followed by P. kelmania, P. pini, and P. cambivora. These trends were not absolute; for example, P. pini was quite virulent on the more susceptible host species, and statistical analysis indicated that significant differences between mean root rot severity caused by P. cinnamomi, P. kelmania, and P. pini were generally perceptible only on resistant hosts (Figure 6). On more resistant hosts (Turkish and Nordmann firs), root rot severity caused by P. kelmania, P. pini, and

P. cambivora rarely deviated statistically from root rot ratings assessed on control seedlings

(Figure 6). Phytophthora cambivora was arguably the least damaging Phytophthora, differing significantly from the other three treatments in nearly every fir species except for noble fir.

Although not comparable statistically, visual observation of root rot damage inflicted by different isolates of P. cinnamomi suggests that Isolate 1 of this species caused less severe damage than the other two isolates tested, particularly at cool temperatures and on more resistant hosts. Such a trend was not immediately perceived among isolates of the other three

Phytophthora species, although mathematical analyses were not performed to substantiate this observation.

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Disease Progress. Disease progress curves representing mortality over time and mean separations among mortality percentages for each fir species under each treatment are illustrated in Figure 8 and Table 4, respectively. Mortality was generally lower in the cooler greenhouse than under warmer ambient temperatures. Phytophthora cinnamomi generally affected a greater number of species than other Phytophthora treatments. Phytophthora kelmania and P. pini had little effect on Nordmann and Turkish firs, but were moderately-to-highly virulent on the other hosts, particularly at warmer temperatures. Phytophthora cambivora generally only damaged noble and Fraser firs, with some moderate effect on white fir.

Mean mortality of noble and Fraser firs often clustered statistically, displaying greater sensitivity than other hosts (Table 4). As with other response variable analyses, balsam and white fir were conditionally sensitive. Analysis of mortality in these two species indicated occasional commonality with both susceptible and resistant hosts; but mean separations also showed that these species tended to be similar to each other, displaying statistical independence from all other hosts (Table 4). Canaan fir was generally more resistant and only rarely showed statistical independence from Nordmann and Turkish firs (Table 4).

Radial Growth of Phytophthora Cultures at Two Temperatures. Replicated radial growth assays of all isolates of the Phytophthora species used in this experiment indicated that colony expansion on solid media tended to progress at a higher rate at 27°C than 15°C, although the differences were not statistically significant in some isolates (Figure 9).

Discussion & Conclusion.

Assessments of aggressiveness among the different Phytophthora isolates collected for this study suggested that cultures obtained from different hosts and geographical areas varied little in their abilities to cause lesions on noble fir seedling stems under the experimental

47 conditions. It is postulated that sample sizes and geographical breadth may influence the ability to observe large variations; for example, P. cinnamomi samples were collected exclusively from farms in northern California and North Carolina where this pathogen is historically prominent

(Benson and Grand 2000; Wunderlich and Chastagner 2008). Similarly, isolates of P. cambivora were exclusively collected from farms in western Washington and Oregon where this pathogen was found to be causing the bulk of PRR in noble fir (McKeever and Chastagner 2016). Isolates of P. kelmania and P. pini represented slightly greater ecological scope, having been collected from a greater variety of hosts and locations, but the isolates tested from each of these species still varied little in their ability to cause lesions on noble fir stems. In contrast, observational variation between isolates of each Phytophthora species utilized in the greenhouse inoculations indicate that differences may have existed in respective virulences among isolates; although this was not statistically comparable due to the design of the study. For example, Phytophthora cinnamomi Isolate 1 produced statistically lower lesions on noble fir stems than other isolates during virulence testing, which translated to lower root rot severities, slightly higher biomass measurements, and noticeably slower disease progress on hosts in the greenhouse screening.

Parallels between Phytophthora virulence on host stems and aggressiveness on roots are previously established (Weiland et al. 2010), and observed variation among isolates of a given

Phytophthora suggest that virulence testing as a means to characterize pathogen isolates prior to experiment initiation may be an important foundation for Phytophthora inoculation studies.

In conjunction with this idea, the breadth of the origins of collected isolates should also be considered when remarking on the congruency of their virulences. At least one isolate within each species produced statistically smaller lesions than the others tested, suggesting that greater variation may be revealed with larger sample sizes.

48

The contrasting temperatures at which the study was conducted resulted in conspicuous differences in mortality and disease severity in all hosts. In most cases, inoculations performed at warmer temperatures resulted in greater mortality, higher percentages of rotted roots, and larger reductions in dry weight than identical treatments at cooler temperatures. It is speculated that more rapid above-ground mortality in warmer temperatures may be attributable to increased host transpiration rates and a resulting imbalance between moisture loss and the ability of the damaged root system to restore water to the plant, resulting in swift foliar death. Observations of more rapid radial growth of Phytophthora cultures on solid media at warmer temperatures suggests also that the pathogen may have an increased ability to colonize root tissues at higher temperatures, which was reflected in the greenhouse by greater root disease severity and reduced biomass in host roots at 27°C as compared to 15°C. It has been established that P. cinnamomi is well adapted to warm conditions and is a common associate of subtropical plants (Erwin and

Ribeiro 1996). In vitro radial growth studies with P. kelmania (unpublished) have also indicated the relative fitness of this undescribed species at warmer temperatures. Phytophthora cambivora is typically regarded as being adapted to more temperate conditions (Erwin and Ribeiro 1996), and minor differences in root rot severity and dry weight reductions between the two greenhouses with the P. cambivora treatments may have been reflective of this adaptation.

Despite these observations, mortality of noble and Fraser firs caused by P. cambivora still appeared to be slightly larger at 27ºC than 15°C (Table 4). Phytophthora pini is a member of the

P. citricola complex; of which, species are more typically regarded as cohorts of temperate soils;

(Erwin and Ribeiro 1996; Hong et al. 2011) however, interpretation of the results of P. pini radial growth assessments and virulence on Abies hosts at disparate temperatures support the

49 observation that warmer temperatures augment virulence and radial expansion of this species as well.

To address the relevance of this study to molecular marker development and the potential use of genomic tools for Abies improvement, it is important to discuss how host population resistance phenotype distributions may be dependent on the conditions implemented in the inoculation studies. Analysis of the ratio of resistant-to-susceptible individuals following an inoculation provides insight into the number of genes conferring resistance (Flor 1971) which facilitates subsequent efforts for chromosome mapping and estimating the magnitude of the influence of each contributing gene. The present study demonstrated that isolate choice and temperature impact the distributions of the host resistance phenotypes, which may result in uncertainty about whether resistance is being conferred by a single major gene or by numerous cooperating genes. For example, when assessing the extremes of Abies-Phytophthora combinations; that is, a highly susceptible host paired with a highly virulent pathogen or a resistant host with a weak pathogen, there is little resolution for observing gene contributions because one will observe either an excess or dearth of mortality, respectively. The manipulation of temperature or isolate virulence in such an instance could result in alternative host phenotype distributions and subsequent misinterpretation of gene contributions. To accommodate the influences of external factors on host symptom expression, it may be valuable to incorporate variability and intensify replication to assist with clarification of results.

In the present study, root rot severity and root biomass were also assessed in addition to host mortality for determining relative host resistance or susceptibility. Strong correlations among the three response variables indicate that these parameters suitably infer the same observable trends, allowing any of the three variables to be used to gather information about

50 interactions between the different hosts and pathogens. However, when conjecturing about the ability to predict disease progress based on the extent of root rot or existing root biomass, linear regression indicated that only root rot severity is significantly related to host mortality and rapidity of death; whereas root biomass assessments were inadequate for forecasting AUDPC. It is postulated that although individual roots may become decomposed, the structural integrity of the tissue could perhaps be sustained enough to result in only small reductions in weight, particularly over only a 16 week exposure. Correspondingly, secondary regeneration of healthy roots over damaged roots has been observed in certain conifer species including Douglas-fir

(Pseudotsuga menziesii) and Canaan fir, which may render measurements of weight inadequate for predicting host mortality (Frampton 1999; Hansen et al. 1980). It may even be argued that an abundance of fungal tissue in a colonized root may contribute to overall mass, leaving measurements of root weight disputable, but no studies have been performed to quantify this postulation. Anecdotal observations by the authors of grave reductions in root biomass of severely rotted root systems in comparison to healthy ones suggest that the assessments of root biomass conducted in this study may just not have provided enough sensitivity to detect small biomass changes resulting from moderately-susceptible interactions. It is possible that differences in remaining root biomass may only be perceptible in cases where a host-pathogen interaction results in severe rot and ensuing tissue reduction.

Conclusion

The observed interactions among hosts, pathogen, and environment confirmed prior knowledge of the complexity of the PRR-Abies pathosystem and complimented previous research that indicates a lack of resistance in species such as Fraser and noble firs. Clarifications of how Phytophthora species and temperature affect host symptom expression may help to

51 influence interpretation of ostensibly conflicting PRR research emerging from discrete geographic areas by acknowledging that the spectrum of resistances among species of Abies should be considered individually for each unique region. Implementation of a multifactorial approach illuminated the importance of Phytophthora isolate virulence and documented the contribution of ambient temperature to disease development. Knowledge of how temperature influences physiology and pathogen function may assist nursery managers and growers to manipulate environment as a means of disease avoidance, and delineation of the conditions under which more moderately-resistant Abies species are apt to thrive may increase their utility as

Christmas trees in lieu of highly susceptible firs. It is recommended that researchers pursuing studies of genomic disease resistance to PRR understand external influences on host reactions and apply appropriate variation during experimental design to ensure that molecular tools are applicable across a broad range of conditions.

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Chapter 2. Literature Cited

Beck, D.E. 1990. Abies fraseri (Pursh) Poir., Fraser fir. In Burns, R. M. and B. H. Honkala

(Eds.). 1990. Silvics of North America, Volume 1, Conifers. U.S. Department of

Agriculture, Forest Service, Agriculture Handbook 654, Washington, D.C. Pp. 675.

Benson, D.M., Grand, L.F. 2000. Incidence of Phytophthora root rot of Fraser fir in North

Carolina and sensitivity of isolates of Phytophthora cinnamomi to metalaxyl. Plant

Disease 84: 661-664.

Benson, D.M., Hinesley, L.E., Frampton, J., Parker, K.C. 1997. Evaluation of six Abies spp. to

Phytophthora root rot caused by Phytophthora cinnamomi. Biological and Cultural Tests

for Control of Plant Diseases 13: 57.

Bhat, R. G., Colowit, P. M., Tai, T. H., Aradhya, M. K., and Browne, G. T. 2006. Genetic and

pathogenic variation in Phytophthora cactorum affecting fruit and nut crops in California.

Plant Disease 90:161-169

Campbell, S.J., and Hamm, P.B. 1989. Susceptibility of Pacific Northwest conifers to

Phytophthora root rot. Ornamentals Northwest Archives 13(4): 5-8.

Campbell, C.L. and Neher, D.A. 1994. Estimating disease severity and incidence. Pp. 117-147.

In Campbell, C.L. and D.M. Benson (Eds.). 1994. Epidemiology and Management of

Root Diseases. Springer-Verlag: Berlin Heidelberg. 344 pp.

Chastagner, G. A., and Benson, D. M. 2000. The Christmas tree: traditions, production, and

diseases. Online. Plant Health Progress doi:10.1094/PHP-2000-1013-01-RV.

Chastagner, G.A., Riley, K.L., Hamm, P.B. 1990. Susceptibility of Abies spp. to seven

Phytophthora spp. Phytopathology 80: 887.

Eggers, J.E., Balci, Y., MacDonald, W.L. (2012). Variation among Phytophthora cinnamomi

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isolates from oak forest soils in the eastern United States. Plant Disease 96(11): 1608-

1614.

Erwin, D.C. and Ribeiro, O.K. 1996. Phytophthora Diseases Worldwide. St. Paul, Minnesota:

APS Press. 562 pp.

Flor, H.H. 1971. Current status of the gene-for-gene concept. Annu. Rev. Phytopathol. 9: 275 –

296.

Frampton, J. 1999. Evaluating alternative fir species for Phytophthora Root Rot resistance.

Limbs & Needles 26: 7, 10.

Frampton, J. 2005. Exotic fir research in North Carolina. Christmas Trees 32(1): 36-40.

Frampton, J. and Benson, D.M. 2004. Phytophthora root rot mortality in Fraser fir seedlings.

HortScience 39(5): 1025-1026.

Frampton, J., and Benson, D.M. 2012. Seedling resistance to Phytophthora cinnamomi in the

genus Abies. Annals of Forest Science 69(7): 805-812.

Frampton, J., Isik, F., Benson, D.M. 2012. Genetic variation in resistance to Phytophthora

cinnamomi in seedlings of two Turkish Abies species. Tree Genetics and Genomes 9(1):

53-63.

Grand, L.F. and Lapp, N.A. 1974. Phytophthora cinnamomi root rot of Fraser fir in North

Carolina. Plant Disease Reporter 58(4): 318-320.

Granke, L.L., Quesada-Ocampo, L.M., Hausbeck, M.K. (2011). Variation in phenotypic

characteristics of Phytophthora capsici isolates from a worldwide collection. Plant

Disease 95(9): 1080-1088.

Hamm, P.B.; Hansen, E.M. 1982. Pathogenicity of Phytophthora species to Pacific Northwest

conifers. European Journal of Forest Pathology 12(3):167-174.

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Hansen, E.M., Roth, L.F., Hamm, P.B., Julis, A.J. 1980. Survival, spread, and pathogenicity of

Phytophthora spp. on Douglas-fir seedlings planted on forest sites. Phytopathology 70(5):

422 – 425.

Hinesley, L.E., Parker, K.C., Benson, D.M. 2000. Evaluation of seedlings of Fraser, Momi, and

Siberian fir for resistance to Phytophthora cinnamomi. HortScience 35(1): 87-88.

Hodgson, W.A. and Sharma, K.P. 1967. Restoration of virulence of stored cultures of

Phytophthora infestans. Canadian Journal of Plant Science 47: 447-449.

Holmes, K.A., Benson, D.M. (1994). Evaluation of Phytophthora parasitica var. nicotianae for

biocontrol of Phytophthora parasitica on Catharanthus roseus. Plant Disease 79(2): 193-

199.

Hong, C., Gallegly, M.E., Richardson, P.A., Kong, P. 2011. Phytophthora pini Leonian

resurrected to distinct species status. Mycologia 103(2): 351 – 360.

Horsfall, J.G. & Barratt, R.W. (1945). An Improved Grading System for Measuring Plant

Disease. Phytopathology 35: 655.

Kuan, T.L. and Erwin, D.C. (1980). Predisposition effect of water saturation of soil on

Phytophthora root rot of . Phytopathology 70(10): 981-986.

Madden, L. V., Hughes, G., van den Bosch, F. 2007. The Study of Plant Disease Epidemics.

APS Press: St. Paul, MN. 432 pp.

McKeever, K.M. and Chastagner, G.A. 2016. A survey of Phytophthora species associated with

Abies in U.S. Christmas tree farms. Plant Disease 100(6): 1161-1169.

Rhoades, C.C., Brosi, S.L., Dattilo, A.J., Vincelli, P. (2003). Effect of soil compaction and

moisture on incidence of Phytophthora root rot on American chestnut (Castanea dentata)

seedlings. and Management 184: 47-54.

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Shew, H.D., and Benson, D.M. 1981. Fraser fir root rot induced by Phytophthora citricola. Plant

Disease 65: 688-689.

Weiland, J.E., Nelson, A.H., Hudler, G.W. 2010. Aggressiveness of P. cactorum, P. citricola I,

and P. plurivora from European beech. Plant Disease 94(8): 1009 – 1014.

Wunderlich, L. and Chastagner, G.A. 2008. Challenges to managing Phytophthora root rot in

California’s Sierra Nevada foothills. Page 42 in: Proceedings of the 8th International

Christmas Tree Research and Extension Conference. Thomsen, I.M., Rasmussen, H.N.,

Sørenson, J.M. (Eds.), August 12-18, 2007. Bogense, Denmark. 146 pp.

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Chapter 3. Field Assessment of Turkish fir (Abies bornmuelleriana) Resistance to Five

Root-Rotting Phytophthora Species

Employing host resistance to manage Phytophthora Root Rot (PRR) is an active area of research; however, delineating true PRR host resistance is complicated by the variety of Abies species that serve as hosts, the array of Phytophthora species that can cause disease, and inconsistencies in symptom expression under variable regional climates. Efforts to identify resistance in Fraser and noble firs have demonstrated that there is little natural resistance in these species (Frampton and Benson 2004; Frampton et al. 2003; Hinesley et al. 2000); however, it has been established that there is a spectrum of resistance to PRR among the other various species of

Abies that have been considered for Christmas tree production (Talgø and Chastagner 2013).

A variety of different exotic firs have been investigated as alternatives to the common

North American firs. Momi fir (A. firma Siebold & Zucc.) and West Himalayan fir (A. pindrow

(Royle ex D. Don) Royle) have demonstrated excellent survivability in greenhouse inoculation trials (Benson et al. 1997; Hinesley et al. 2000; Frampton and Benson 2012); however, these species tend to make poor Christmas trees due to unattractive form, bristly foliage, and unrefined branching structure (Frampton et al. 2013). King Boris fir (A. borisii-regis Mattf.), Cicilian fir

(A. cilicica (Ant. & Kotschy) Carriére), Turkish fir (A. bornmuelleriana Mattf.), Trojan fir (A. equi-trojani (Asch. & Sint. ex Boiss) Mattf.), Nordmann fir (A. nordmanniana (Steven) Spach.), and Siberian fir (A. siberica Ledeb.) are considered intermediately resistant to PRR, performing slightly better than intermediately susceptible North American Abies species including grand fir

(A. grandis (Douglas ex D. Don) Lindley), balsam fir (A. balsamea (L.)Mill.), Canaan fir (A. balsamea var. phanerolepis Fern.) and white fir (A. concolor (Gordon) Lindley ex Hildebrand)

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(Campbell and Hamm 1989; Frampton and Benson 2012; Hinesley et al. 2000; Hamm and

Hansen 1982).

Decisively characterizing the long-term resistance potential of these intermediate trees is complicated by a number of interacting factors including external stressors that affect symptom development (ambient temperature, status) as well as which species of Phytophthora are responsible for causing disease. Variation in aggressiveness among Phytophthora spp. and geographic variability of Phytophthora community compositions can result in disparate research results from different geographic zones. For example, Turkish and Nordmann firs are widely considered to be reliably resistant to PRR in the Pacific Northwest where P. cambivora, P. cryptogea, or P. plurivora Jung & Burgess are oft encountered, but these species fail more commonly in growing regions in California and North Carolina where P. cinnamomi is the predominant PRR-causing Phytophthora (Chastagner and Benson 2000; Frampton et al. 2012;

Wunderlich and Chastagner 2008). Similarly, greenhouse inoculation studies in the Pacific

Northwest have demonstrated less than 30% mortality of white and balsam firs when challenged with P. cambivora and P. pini (unpub), while studies in North Carolina have demonstrated nearly 100% mortality of these species when inoculated with P. cinnamomi (Frampton and

Benson 2012). These conflicting conclusions can complicate disseminated information regarding perceived host resistance and recommendations for host species substitutions; particularly when considering Abies spp. that display intermediate or variable reactions to PRR (Hamm and Hansen

1982).

In recent decades, the search among exotic firs for a PRR-resistant Christmas tree has narrowed its focus toward Nordmann, Turkish, and Trojan firs because of their observed tendency to remain healthy in infested areas and horticultural characteristics that make them

58 attractive as a Christmas tree. The native range of Nordmann fir extends from the Caucasus

Mountains of Russia and the Republic of Georgia, westward toward the Black Sea region of northern and western Turkey where it intersects the native ranges of Turkish and Trojan firs.

These three species are closely related and are commonly referred to as the

Species Complex (Kaya et al. 2008). Nordmann fir is the most popular Christmas tree in Europe, with upwards of nine million trees produced annually in Denmark alone (Cregg and Bates 2007,

Nielsen and Chastagner 2005). They are praised as having excellent conical form, polished green foliage, and an open branching habit (Cregg and Bates 2007). Their plasticity to tolerate a wide range of soil pH conditions facilitates successful cultivation in a variety of geographic regions.

Certain sources of Nordmann fir have been demonstrated to exhibit good postharvest needle and moisture retention, which is an important characteristic for U.S. markets where Christmas trees are increasingly maintained for weeks in the home subsequent to harvest (Chastagner and Riley

2003; Nielsen and Chastagner 2005). Research to identify seed sources that combine superior germination, seedling height, bud characteristics, and improved postharvest quality has been carried out to further refine the usefulness of these species in the U.S. Christmas tree industry

(Nielsen and Chastagner 2005; Sevik 2012). Other studies have indicated their relative tolerance to a variety of conifer pests including balsam woolly adelgid, annosus root rot, and spider mites

(Chastagner et al. 2003; DeFrancesco and Murray 2009; Newton et al. 2012; Pscheidt and

Ocamb 2015).

Although trees in the Abies nordmanniana complex are regarded as exhibiting an overall resistance to PRR in most growing regions, incongruences in research results and a higher rate of failure in areas with P. cinnamomi indicate that all seed sources may not be uniformly resistant.

A 1997 lath house inoculation study using P. cinnamomi showed high survival and low foliar

59 disease ratings of multiple sources of Nordmann and Turkish fir despite high isolation success from inoculated roots (Benson et al. 1997). In contrast, a similar 2012 study observed mortality levels of 61.3% among an undisclosed number of Turkish fir sources, 77% among multiple

Nordmann fir sources, and as high as 84.2% in Trojan fir (Frampton and Benson 2012). To further understand the dynamics of resistance to P. cinnamomi among seed sources of these exotic firs, these same researchers initiated a subsequent study to test the survival of 34 open- pollinated families of Trojan fir and 71 families of Turkish fir. These trials indicated enormous variation in mortality among seed sources, with mortality ranging from 19.7% to 86.5% between

Trojan fir families and from only 6.4% mortality to as much as 82.8% mortality among families of Turkish fir (Frampton et al. 2013). Family-level variation was also witnessed in a field trial in

California where eight trees each of three sources of Turkish fir and nine sources of Nordmann fir were outplanted into three Christmas sites that were severely impacted by natural populations of P. cinnamomi (Wunderlich and Chastagner 2008). Focusing on the only one of the three study sites that positively yielded Phytophthora as the causal agent upon isolations from host roots, survival among the three selected seed sources of Turkish fir was consistently low, with only 38% survival (62% mortality) in each. The larger number of Nordmann fir sources at this revealed greater variation, with two stand-out seed sources that showed 50% and

75% survival in comparison to a range of 0-38% survival for the other seven sources

(Wunderlich and Chastagner 2008). These trials reinforce the assertion that resistance to PRR is dependent on host genetics and further research has demonstrated that family mean heritability values are high (0.97) for this phenotypic attribute (Frampton et al. 2013).

In order to study variation in PRR resistance among seed sources of exotic firs, several families of Turkish fir seedlings obtained through the Cooperative Fir Germplasm Evaluation

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(CoFirGE) project (Chastagner et al. 2015; Kurt et al. 2016) were employed in a field trial to assess differences in seed source survival under heavy PRR disease pressure. CoFirGE seedlings selected for this field study were produced from seed that was collected in 2010 from the

Adapazarı, Bolu, and Karabük provenances along an elevation gradient in each sampling site that ranged from 280 m to 1,690 meters (Frampton 2011).

Previous inoculation studies have focused primarily on lath house-based trials in warm, humid climates in the southeastern U.S. using only P. cinnamomi; thus, it was of interest to evaluate resistance under field conditions in the temperate Mediterranean climate of the Pacific

Northwest where 40% of U.S. Christmas tree production is centered, and also to include a wider variety of Phytophthora spp. to assess pathogen aggressiveness and host specificity. To complement an overall evaluation of the seed sources included in this study, trends in resistance/susceptibility along elevation gradients were evaluated and comparisons were drawn between the three provenances of Turkish fir to validate a 2013 study by Frampton et al. in which greater resistance to P. cinnamomi was observed in Turkish fir seed sources from the easternmost provenance.

Materials and Methods

Study Site/Plot

The initial experiment was conducted from May 22 through September 11, 2013 and was repeated from May 25 through September 11, 2014. Both trials were performed at the

Washington State University Puyallup Research and Extension Center (47°11’40”N,

122°19’57”W) in soil classified as a Puyallup fine sandy loam soil (USDA NRCS 2016). Square plots were established each year and were situated approximately 40 m apart to avoid carry-over of inoculum from the first year to the second. Each year’s plot measured 17 x 17 m and was

61 partitioned into 30 replicate blocks (1.8 x 1.2 m). To foster disease development in the study sites in each year, flooding was imposed for a period of time following inoculation so plot construction necessitated a system to establish water flow and maintenance. To achieve this, 0.5 m high berms were constructed around the entire exterior of the outer plot to facilitate water retention. An additional berm was constructed to bisect the plot to aid with ground leveling and to relieve water pressure on the outer berms during flooding. The plot was leveled using a Bosch

GRL400HCK industrial laser levelling system and redistributing soil by hand to create an even grade for maintaining consistent water levels during the flooding period. Polyvinyl chloride

(PVC) outflow pipes were embedded in the berms to prevent breaching and to assist in preserving desired water levels inside the plot. A Washington State University (WSU) weather station located within 400 m of the plot recorded daily temperature highs and lows, relative humidity, , and soil temperature at 20 cm belowground.

Inoculum

Single isolates of each of five Phytophthora species, P. cambivora, P. cinnamomi, P. pini, P. cactorum, and P. cryptogea, were used to produce rice grain inoculum for this study

(Table 1). These five species were selected to comprise a diverse representation of the most pervasive Phytophthora spp. causing disease on Abies in U.S. plantations. Rice grain inocula were prepared following methods developed by Holmes and Benson (1994). Prior to use, viability of inocula were verified and rice grains were pulverized in a kitchen-grade food processor before being screened through a 0.8 mm (#20) soil sieve. 3.4 g portions of each of the five rice grain inocula were mixed together to create a homogenized 17 g mixture for each of the

30 replicate field blocks. To aid distribution of inoculum into the field, each 17 g portion of

Phytophthora inoculum was mixed with 1,700 ml (by volume) of loosely-packed potting mix

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(Gardener’s Professional Secret, Specialty Soils, Covington, WA) to serve as a carrier. Prior to inoculation, field soil was sampled from eight of the replicate blocks to estimate a baseline level of native Phytophthora in the soil using baiting and dilution plate methods (Larkin et al. 1995;

Themann et al. 2002). Near dusk of the day of inoculation, the rice grain inoculum blended in the carrier was hand-shaken into each delineated replicate block and immediately turned into the top

10 cm of soil using a metal garden rake. Infested soil in each block was kept moistened until all blocks were completed. Upon completion of inoculation, the ground was kept wet by periodic overhead sprinkle irrigation to ensure viability of the inocula until planting the following day.

Prior to planting of seedlings into the infested ground, soil in the inoculated blocks was sampled using the baiting and dilution plating methods described above to verify viability of the introduced inoculum.

Seedlings

CoFirGE seedlings were grown as containerized stock for one year at Kintigh’s Mountain

Home Ranch in Springfield, Oregon, prior to packaging and distribution to CoFirGE collaborators in February of 2013. Thirty-six total Turkish fir seed sources composed of 12 families from each of the three Turkish provenances were selected from the collection of

CoFirGE seedlings for testing in this Phytophthora study (Figure 1). Within each set of 12 seedlings representing a provenance, four sources were selected to comprise the low, mid, and high elevation categories of mother trees. Additional seedlings employed in this study included two of the Danish Nordmann fir families and one source each of Fraser and noble firs that were part of the original CoFirGE project. The inclusion of noble and Fraser firs served as susceptible sentinel trees to validate the presence of Phytophthora in the event that the resistant Turkish and

Nordmann firs were slow to display symptoms. The one-year old containerized seedlings were

63 transplanted into 950 ml MT38 Mini-Treepots (Stuewe & Sons, Tangent, OR) at the WSU

Puyallup Research and Extension Center and maintained outdoors for an additional three months prior to initiation of the first trial of the field study. Seedlings to be used for the second trial were maintained for an additional year prior to transplanting to the field site.

Planting

The morning following field inoculation, tagged seedlings were randomized and planted into the moist soils within each of the inoculated replicate blocks. A posthole digger was used to create planting holes and the resultant cores were reused to pack soil back around the planted seedlings to ensure that infested soil was fully in contact with host roots. Each replicate block held 40 seedlings comprising the 36 Turkish fir families, two Nordmann fir families, Fraser fir, and noble fir. Prior to initiation of flooding, seedlings were left undisturbed for seven days to encourage establishment and reduce transplant stress. During this period seedlings were overhead irrigated for 20 min, two times per day, via a center pivot impact sprinkler head mounted on a vertically-oriented 1.8 m irrigation boom that extended from the initial field irrigation system riser.

Flooding

On day seven, flooding was initiated by expulsing water from uncapped irrigation pipe directly into the footprint of the plot. When the floor of the plot was fully submerged, the irrigation flow rate was continuously adjusted to ensure a static level at the lower crown level of the seedlings for an uninterrupted 48 hour submersion period. While flood waters submerged the plot, whole sterilized bait leaves were floated in mesh bags near the outflow pipes of the plot as validation of the presence and viability of Phytophthora inoculum in the water.

Since the subterranean irrigation system that supplies the study site sources water from a nearby

64 creek, aliquots of outflow water were sampled for the presence of irrigation-borne Phytophthora using the rhododendron leaf disc bait method. Upon completion of the flooding exposure, irrigation was discontinued and the water was allowed to percolate naturally through the soil to drain the plot. Rhododendron leaf bait bags were removed and portions of the leaves were surface sterilized and plated into PARPH-cV8 medium, as described above, to recover

Phytophthora.

Overhead irrigation was continued twice daily for 20 min durations for the next several weeks until seedlings broke dormancy and newly-expanded foliage had lignified. Once the new foliage had hardened off, irrigation was discontinued and an overhead application of the pre- emergent oxyfluorfen (GoalTender, Dow AgroSciences, Indianapolis, IN) was applied to assist with weed control. Manual weed control was conducted periodically during the course of the season.

Sampling/Isolation

Visual foliar ratings were conducted weekly to identify trees that were failing (wilting, chlorotic) or dead. Seed source identity and plot position of dead seedlings were noted and dead trees were removed from the plot for recovery of the pathogen. Upon removal, seedling roots were washed free of clinging soil and 10 symptomatic root tips from each tree were surface sterilized, as described above, before plating into PARPH-cV8 semi-selective medium. All resulting Phytophthora isolations were subcultured to un-amended cV8 medium. Culture identities were verified based on morphology and sequencing of the internal transcribed spacer

(ITS) region of the ribosomal DNA. Phytophthora DNA was extracted using the QIAGEN

DNeasy Plant Mini Kit (QIAGEN, Inc., Valencia, CA, USA) following manufacturer’s instructions. ITS regions were amplified using ITS6F and ITS4R primers (Cooke et al. 2000).

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PCR conditions for ITS amplification included initial denaturation at 94°C for 3 min followed by

35 cycles of 30 s at 94°C, 30 s at 55°C and 1 min at 72°C, with a subsequent 10 min at 72°C.

PCR products were prepared for sequencing by mixing 2 µl of ExoSAP-IT (Affymetrix, Santa

Clara, CA) with 5 µl PCR product and following the manufacturer’s thermocycler protocol of 15 min at 37°C and 15 min at 80°C. Prior to sequencing by GENEWIZ (South Plainfield, NJ), diluted 3.125µM forward primer was added to the treated PCR products. For identification, resulting ITS sequences were compared to published sequences in the National Center for

Biotechnology Information (NCBI) GenBank BLASTn database. After ITS sequence-based identification of isolates in the initial stages of the study, latter identifications could be made by morphological examination alone.

Data Analysis

R statistical software (R Foundation for Statistical Computing, Vienna, Austria) was utilized to determine statistical significance of the data derived in this study. Chi-square analysis of the frequency of dead seedlings in each seed source was implemented to provide a guideline for assessing susceptible seed sources.

The Kruskal-Wallis non-parametric one-way analysis of variance (ANOVA) on ranks was calculated to assess differences in overall mortality among the three provenances of Turkish fir, treating the 12 seed sources representing each provenance as replications. Post-hoc multiple comparisons of means were performed using Dunn’s and Nemenyi’s tests to analyze the sample pairs for stochastic dominance. The purpose of this test was to compare the current data with an earlier study in which researchers observed a trend of reduced susceptibility (less mortality) in seed sources derived from the easternmost provenances following lath house inoculations of seedlings with P. cinnamomi (Frampton 2009).

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Due to the low-, mid-, and high-elevation selections of seed sources within a provenance, three separate parametric one-way ANOVAs were performed for each provenance to assess differences in mortality among the three elevation categories. The four seed sources that represented each elevation category within each provenance were treated as replicates. Upon the occurrence of a significant ANOVA test, the Fishers Least Squared Difference (LSD) post-hoc pairwise comparison test was used to assess differences between the three elevation categories.

Phytophthora species aggressiveness was assessed by examining both the proportion of mortality caused by each Phytophthora spp. with respect to host species or provenance, as well as overall percentage of mortality caused by each respective species across all hosts. For the former objective, the total number of isolations of each respective Phytophthora spp. was tallied and divided by the total number of dead seedlings. The latter objective was assessed by tallying the total number of isolations of each Phytophthora from each host species and dividing by the number of dead seedlings for each respective host. Fisher’s LSD hypotheses tests were used to assess differences in host mortality due to individual Phytophthora species.

Results

Environment. Daily air temperatures averaged over the duration of the experiments were 17.6ºC and 17.9ºC for 2013 and 2014, respectively, with the same average high temperature of 24ºC.

Average daily relative humidity was calculated to be 76% in 2013 and 73% in 2014. Soil temperatures at 20 cm below ground averaged 20ºC and 23ºC for years 1 and 2 respectively. In

2013, there were 30 days of measurable precipitation with an average of 0.15 cm rainfall throughout the 113 day period of the experiment. In 2014, there were 20 days of measurable throughout the 110 days of the experiment, with an average of 0.08 cm over all days.

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Symptoms and Baiting. Mortality of trees was evident within three weeks of flooding.

Symptoms included wilting of newly-expanded growth, chlorosis, foliar necrosis, and dieback of branch tips that were in contact with flood waters. Rhododendron leaf baiting and dilution plating of the soil prior to introduction of inoculum yielded no native Phytophthora in the plot areas. Following inoculation, soil sampling and dilution plating allowed recovery of four of the five Phytophthora added to the system. P. pini was not recovered during this post-inoculation soil sampling; however, recovery of this species from rhododendron leaves floated in the flood waters and seedling roots during the experiment verified its presence. Rhododendron leaf baiting of the flood waters provided recovery of only P. cinnamomi and P. pini, but the other species were verified via soil sampling as previously mentioned. Finally, baiting of the irrigation outflow water did not reveal any extemporaneous Phytophthora originating from the unfiltered creek water that served as a reservoir for the field site irrigation system.

Seedling Mortality. Mortality of noble and Fraser fir provided ample contrast to observe the disparity between highly susceptible Abies species and intrinsically more resistant ones such as

Turkish fir. Percent seedling mortality pooled over the two years of the study was 85% for Fraser fir and 75% for noble fir (Figure 1). In contrast, the most susceptible Turkish fir seed source

(#47) still had less than 20% overall mortality. A significant chi square statistic (p<0.001) indicated rejection of the null hypothesis to conclude that all seed sources did not perform uniformly. Families that deviated significantly from the chi-square value included four that suffered mortality in excess of 10% and were thus considered susceptible seed sources. These four sources, #2, #8, #47, and #50, were distributed among the Bolu and Adapazarı provenances

(Figure 1). Of the 32 other, non-significant, families, 72% had mortality percentages of less than

5%, provoking an assertion that most sources included in the study are resistant to PRR.

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Due to the inability to normalize grouped provenance mortality data, the Kruskal-Wallis one-way ANOVA on ranks test was used for determination of differences in mortality across the three provenances of Turkish fir. A significant p-value (p<0.001) compelled rejection of the null hypothesis, indicating that there were indeed differences in susceptibility among the three provenances. Post-hoc multiple comparisons performed using both Dunn’s and Nemenyi’s tests returned very similar results, indicating that the Bolu provenance differed significantly from the

Karabük, but that the Adapazarı provenance did not differ significantly from either the Bolu or

Karabük provenances (Figure 2). A low mean mortality of 1.16 trees in the Karabük provenance indicates that seed sources from this provenance are the least susceptible; while higher mean mortality of 4.08 trees in seed sources from the Bolu provenance indicate that, in this study, this was the most susceptible provenance.

Within each provenance, seed sources were grouped with respect to three elevation categories from which their mother trees stood. The three elevation categories (1,030 to 1,200 m;

1,230 to 1,400 m; and 1,430 to 1,700 m) were compared to survey differences in overall mortality. There were no significant differences in susceptibility of the seed sources representing each elevation grouping in either the Bolu or the Karabük provenances; however, analysis of variance of the mortality among elevations in the Adapazarı provenance was significant

(p=0.007). Fisher’s LSD indicated that the low elevation range was significantly different from either the moderate or high elevation categories. There was greater mean mortality of 6.75 trees among seedlings in the low elevation range of the Adapazarı provenance, compared to a mean of

2.25 trees in the mid elevation and 0.75 trees in the high elevation, indicating that the low elevation sources were more susceptible (Figure 3).

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Phytophthora recovery. Phytophthora cryptogea was recovered from every Abies species employed in this study, including each provenance of Turkish fir (Figure 4). Similarly, nearly every host species was colonized by P. cinnamomi, with the exception of Fraser fir. Noble fir was the only species to host all five species of Phytophthora. When viewing the percent recovery of each Phytophthora spp. pooled over all hosts, regardless of host species, a more than two-fold increase in recovery of P. cryptogea over the next most-abundant species (P. cinnamomi) dictated rejection of the null hypothesis to conclude that there were significant differences in recovery among the five Phytophthora spp. (Figure 5). Phytophthora cinnamomi caused one- fifth of the overall mortality, while the other three species caused only marginal damage in a few hosts.

Discussion.

The overall resistance of both Turkish and Nordmann firs to PRR was established via comparison to the mortality suffered by traditionally more susceptible species such as noble and

Fraser firs. This supports previous proposals that these exotic firs provide options for reclamation of PRR-afflicted fields that may be unsuitable for Fraser and noble fir production. These results suggest that, in areas that harbor P. cinnamomi or P. cryptogea some losses may be expected; but with proper site stewardship and water management, Turkish or Nordmann fir plantings should be more successful than cultivation of other highly susceptible Abies species.

In the context of circumventing PRR by cultivating resistant host species, mortality in excess of 10% may prove to be economically prohibitive for growers, suggesting that the

Turkish fir seed sources that exhibited that level of susceptibility may be inappropriate for use.

The observation that the four less-resistant seed sources were distributed across the Bolu and the

Adapazarı provenances implies that collection of seed from these provenances may pose greater

70 risk for rearing PRR-susceptible trees. There are limited phenotypic characteristics that stand out as explanatory or predictive of the inadequate performance of these four sources; however, characterization of their respective parent trees by the seed collection team indicates that these sources were frequently the progeny of trees growing less optimally compared to other collection trees. Mother trees that produced sources #2, #8, and #50 were rated as having only average vigor on a 1 – 5 scale based on height, leader growth, and branch density relative to neighboring trees. Source #47 had the highest amount of mortality of all 36 sources tested and was derived from the only mother tree in the Adapazarı collection that was evaluated to have poor vigor; a rating of 1 out of 5. As vigor may be an important determinant of the ability to resist disease, lack thereof may be a contributing factor to the observed sensitivity to PRR in these sources.

Data from the current field assessment indicate significantly greater mortality in seed sources from mother trees in the low elevation category of the Adapazari provenance; and although not statistically significant, a higher mean mortality was observed with the low elevation sources of the Bolu provenance (Figure 3). Mother trees that produced #2, #47, and

#50 all stood in the low elevation categories within their respective provenances, and the mother tree for source #8 stood on the lower cutoff of the mid-elevation category at just near 1,230 m.

At lower elevations, Turkish fir are often found intermixed with Oriental beech (Fagus orientalis

Lipsky) and may share canopy dominance or be suppressed by the larger, more quickly growing beech trees (Kurt et al. 2016; Özel and Ertekın 2012). In an intermixed forest type, competition with a rapidly growing, densely-colonizing hardwood species may dictate energy dispensation in favor of height growth and photosynthetic development in lieu of vigorous root growth and disease resistance (Bai et al. 2015).

71

Lath house inoculations of a similar collection of Turkish fir families by Frampton et al.

(2013) indicated an easterly trend of reduced susceptibility to P. cinnamomi among trees from the three provenances, such that sources from the easternmost Karabük provenance had less mortality than those from Bolu; and likewise, sources from the Bolu provenance had less mortality than those from the westernmost Adapazarı (Akyazı province) (Figure 6). Results from the present field experiment using several Phytophthora spp. indicate a similar trend, with significantly less overall mortality among sources in the Karabük provenance as compared to the

Bolu or Adapazarı families. In this study, the trend is not an absolute parallel to the results of

Frampton et al. since the westernmost Adapazarı had slightly fewer dead trees on average than the Bolu; however, the disparity between these two values is statistically null. Concurrent with the conclusions by Frampton et al., it is unknown why susceptibility appears lower in easternmost provenances; but it is surmised that the trend may be a result of co-evolutionary relationships between Phytophthora and Turkish fir in the individual forests, or perhaps due to climate or site characteristics including rainfall, temperature, or soil properties. Summarization of the quality and growth characteristics of the mother trees by the seed collection team show that trees in the Karabük provenance consistently rated higher for qualities such as crown score and color than those from either of the other two provenances; were taller on average than trees in the Adapazarı or Bolu, and produced seed with slightly greater germination success at four weeks (Kurt et al. 2016). Additionally, height and survival measurements of Turkish fir families in the CoFirGE common garden trials indicate that trees from the Karabük provenance are exhibiting more robust survival and are growing taller in comparison to trees from westerly provenances, and that increased survival is correlated with higher altitude seed sources (R2=0.30)

(Frampton, unpublished). Although these obervations appear to endorse the association of tree

72 vigor with disease resistance, regression of CoFirGE height and first-year survival against PRR disease resistance was not significant (data not shown), cautioning that PRR mortality is not likely predictable using these parameters.

Disparities in the mortality caused by each of the 5 Phytophthora spp. that were dispatched as inoculum defied expectations of the authors. Prior work has indicated widespread

Abies infection caused by P. cambivora in fields in western Washington and P. cinnamomi has been established as a formidable pathogen to several species of Abies (Benson et al. 1997;

Frampton and Benson 2004, 2012; McKeever and Chastagner 2016). P. pini and P. cryptogea were recovered recently from field-grown Turkish fir in North Carolina, and P. cactorum has been reported to be severe on fir in the Great Lakes region of the United States (McKeever and

Chastagner 2016; Anette Phibbs per. com).

Despite intrinsic variations in virulence, it was expected that the highly conducive conditions artificially created to promote disease in this plot would provide all five species opportunity to cause disease on Turkish fir, but that P. cinnamomi may burgeon due to the warm soil temperatures typical during the months in which the field studies were executed.

Furthermore, recent results of a controlled lath house inoculation study performed by researchers in North Carolina indicated superior aggressiveness of P. cinnamomi over P. cryptogea on

Turkish and Trojan fir seedlings produced from CoFirGE seed, reinforcing the idea that P. cinnamomi may stand out (Kohlway et al. 2015). The dominance of P. cryptogea in both years of this study, then, disrupted preconceived theories.

When Phytophthora spp. recovery from this study is broken down by host species (Figure

4), it is revealed that more than 70% of both Fraser and Nordmann fir mortality, and at least 50% of the mortality of any of the species/provenances tested was due to P. cryptogea. The failure to

73 recover P. cryptogea during pre-inoculation baitings of the soil and irrigation water dismiss the possibility for native baseline populations of this species in the plot area. Correspondingly, a

2011 Phytophthora sampling effort of the creek water in locations upstream and downstream from the field plot demonstrated recovery of P. gonapodyides (Petersen) Buisman, P. siskiyouensis Reeser et E.M. Hansen, P. megasperma Drechsler, P. lacustris Brasier, Cacciola,

Nechwatal, Jung & Bakonyi, and P. chlamydospora Brasier et E.M. Hansen; but do not indicate presence of P. cryptogea (unpub). The abundance in recovery of P. cryptogea from Turkish fir in this study then implies that a level of specificity may exist between this host and pathogen combination; however the scope of the present data only allows inference that P. cryptogea was also recovered abundantly from other host species, and that Turkish fir was also observed to support four of the five Phytophthora spp. included in the study.

When assessing mortality pooled over all hosts irrespective of host species (Figure 5), one can observe that P. cryptogea and P. cinnamomi caused the majority of disease, with the three less-virulent pathogen species constituting only about 10% of overall mortality.

Interestingly, P. cinnamomi was not recovered from any of the Fraser fir seedlings in both years of the study, which was considered unusual with respect to the historical antagonism of Fraser fir by P. cinnamomi in growing regions in the southeastern United States (Frampton and Benson

2004, 2012). It was also surprising that the extended period of flooding would not favor greater success of the three less successful Phytophthora spp., given copious sporangia production by P. cactorum observed on solid media and the abundant recovery of P. cambivora from fir during a recent survey of western Washington Christmas tree farms (McKeever and Chastagner 2016).

The absence of an non-inoculated treatment in this field study may raise concern over the possibility of seedling mortality as a result of anaerobic conditions in the root zone rather than

74 root damage due to Phytophthora infection; however, prior literature has investigated the predisposing effects of flood exposure to Abies, finding no discernable antagonism by oversaturation of non-inoculated roots for up to a 48 h period (Kenerley et al. 1984). This study concluded that apparent increases in host mortality commonly observed in flooded Phytophthora inoculation studies is due to water-induced provocation of zoospore production rather than an anoxia-induced disruption of root function. Furthermore, an unpublished study performed at

Washington State University that is similar in scope and location to the present study indicated the loss of only a single Fraser fir seedling among 20 non-inoculated plots that were flooded multiple times over the course of five months. Additional studies have corroborated these findings on other hosts including rhododendron and (Blaker et al. 1981; Browne &

Mircetich 1988; Grünwald et al. 2008).

Conclusions

This assessment of Turkish fir resistance to PRR is a pivotal step in determining its potential as an alternative Christmas tree species, as markets shift to adopt more exotic firs.

Variability in PRR resistance of Turkish fir families was validated; however, it was established that Turkish fir still exhibits a level of resistance that is superior to noble and Fraser firs when challenged with high Phytophthora inoculum loads under saturated field conditions. The data suggest that superior family lines may be retrieved from higher elevations in the easterly provenances of the native range of Turkish fir. Aside from PRR resistance, other cultivation and marketability characteristics including optimal bud-break timing, cold acclimation, and superior post-harvest needle and moisture retention are being characterized in an effort to define Turkish fir’s regional adaptability.

75

In the Pacific Northwest, the acceptance of Turkish and Nordmann fir as practical

Christmas trees by both growers and consumers is evidenced by increases in sales, production, and the acquisition of premium prices for these trees. As U.S. markets expand and diversify, it is evident that inherent aesthetic quality and disease resistance will ensure that Turkish fir will remain an economically-important contender for the Christmas tree industry.

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APPENDIX: Chapter 1. Figures and Tables

Figure 1. Maximum Likelihood tree using rDNA ITS sequences showing phylogenetic relationships between Phytophthora isolates collected during this survey and reference Phytophthora sequences obtained from GenBank. Numbers near nodes represent the percentage of trees in which the associated taxa clustered together during bootstrap analysis performed with 1,000 replications.

aThe subset of isolates represented in the phylogeny included at least one isolate per Phytophthora species from each host in each state. Δ Reference sequences for the ITS 1 region from validated isolates that have been accessed via GenBank from the World Phytophthora Collection (WPC) or the American Type Culture Collection (ATCC).

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Table 1: Phytophthora spp. isolated from Abies tissue or associated soils at Christmas tree farms in 9 U.S. states. No. of GenBank Phytophthora spp. Host Statea isolatesb Samplec accession # d P. cactorum Abies balsamea WI 1 Collar N/Se * Abies fraseri CT 2 Root KU053237 KU053236 Abies fraseri MI 2 Soil AY995343 * AY995357 * P. cambivora Abies fraseri MI 1 Soil N/S * Abies procera OR 13 Stem KU053250 KU053251 KU053252 KU053253 KU053254 KU053255 KU053256 KU220617 KU220618 Abies procera WA 20 Stem/Collar KU053259 KU053260 KU053261 KU053262 KU053263 KU053264 KU053260 KU053266 KU053267 KU053268 KU053269 KU053270 KU053271 P. cinnamomi Abies nordmanniana CA 3 Root KU053226 KU053227 Abies equi-trojani CA 2 Root KU053228 Abies equi-trojani NC 2 Root/Stem KU220612 Abies concolor CA 4 Root KU053229 Abies bornmuelleriana NC 2 Stem KU220613 Abies fraseri NC 1 Root N/S * P. cryptogea Abies fraseri NC 2 Stem KU053246 P. europaea Abies fraseri WI 1 Collar N/S * P. gonapodyides Abies grandis OR 2 Branch KU053257 KU053258 P. taxon ‘kelmania’ Abies balsamea var. WI 1 Collar N/S * phanerolepis

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Abies fraseri NY 5 Root KU220615 KU220616 KU053242 KU053244 Abies fraseri CT 4 Root KU053233 KU053234 KU053235 Abies bornmuelleriana NC 7 Root KU053247 Stem KU220614 Abies fraseri NC 6 Stem KU053249 P. megasperma Abies grandis ID 2 Root KU053238 KU053239 Abies fraseri MI 1 Soil AY995367 * Abies procera WA 2 Root KU053272 KU053273 P. nicotianae Abies fraseri MI 1 Soil AY995347 * P. pini Abies balsamea var. CT 1 Root KU053230 phanerolepis Abies fraseri NY 3 Root KU053240 KU053241 Abies fraseri NC 4 Stem KU053248 P. plurivora Abies fraseri MI 1 Soil AY995356 * Abies fraseri WI 1 Collar N/S * Abies fraseri CT 2 Root KU053231 KU053232 P. pseudosyringae Abies fraseri WA 2 Root KU053274 KU053275 P. sansomeana Abies fraseri NY 2 Root KU053243 KU053245 Abies fraseri WI 1 Collar N/S * Abies fraseri MI 1 Soil AY995366 * aU.S. state where isolate was collected bTotal number of isolates collected from a particular Abies spp. in a particular state. cPart of the tree from which a given culture was isolated. Collar = basal bole tissue above and adjacent to the soil line; Root = below-ground root tissue; Stem = bole tissue above the collar; Soil = soil immediately surrounding bole/roots of tree. Tissue samples were obtained from lesion margins demarcating diseased and healthy tissues. dGenBank accession numbers for sequenced isolates. Not all isolates from the total number collected were sequenced. Many isolates were identified based on morphology to others collected in the same area or from the same tree. e N/S, not submitted *, culture provided from collaborating researcher

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Table 2. Pathogenicity of Phytophthora spp. found on Abies hosts for the purpose of establishing new disease reports for various locations.

Avg. Root First Report GenBank Abies spp. Phytophthora spp. Rot Ratinga Locationb Accessionc A. fraseri P. cactorum 3.1 CT KU053237 P. cryptogea 4.0 United States KU053246 P. kelmania 4.0 CT KU053233 4.0 NY KU053242 P. plurivora 4.0 CT KU053232 P. pini 4.8 NC KU053248 4.0 NY KU053241 P. sansomeana 4.0 NY KU053245 A. grandis P. megasperma 3.0 United States KU053238 A. equi-trojani P. cinnamomi 3.0 United States KU220612 A. nordmanniana P. cinnamomi 3.0 CA KU053226 A. balsamea var. phanerolepis P. pini 2.0 United States KU053230 A. bornmuelleriana P. cinnamomi 2.0 United States KU220613 P. kelmania 2.0 United States KU053247 aFive replicate seedlings for each Abies-Phytophthora combination were inoculated and grown in a greenhouse for 8 weeks, at which time the extent of root rot was rated by teasing apart the washed roots and visually estimating the proportion of necrotic root tips on a scale of 0 to 5, where 0 = < 10%, 1 = 10% to 25%, 2 = 25% to 50%, 3 = 50% to 75%, 4 = 75% to 90%, and 5 = > 90% necrotic roots. Two trials were performed. The number listed here is the average root rot rating of the 5 replicate seedlings over two trials. bLocations for first reports established via Koch’s Postulates verification of Phytophthora-Abies interactions observed in this survey. Information on existing first reports was obtained from the Systematic Botany and Mycology Laboratory (SBML) fungus-host database (http://nt.ars- grin.gov/fungaldatabases/fungushost/fungushost.cfm). cGenBank accession numbers for isolates used in these experiments.

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APPENDIX: Chapter 2 Figures and Tables

Figure 1. Mean lesion sizes produced on noble fir seedling stems 7 days post-inoculation with various isolates of 4

Phytophthora species. Isolate identities and states of origin are listed along the x-axis for each Phytophthora species.

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a Letters above bars indicate significantly different lesion sizes produced by the various isolates within a Phytophthora species, as determined by Tukey’s HSD post-hoc analysis.

Figure 2. Relationship between log-transformed area under the disease progress curve (AUDPC) values and root rot rating (expressed as the midpoint value of the percentage range) for each host species. Values for all isolates from 15°C and 27°C are combined on the figures to assess overall relationship between root rot severity and AUDPC. Trendlines represent regression between the variables. BAL = balsam fir, CAN = Canaan fir, FRA = Fraser fir, NOB = noble fir, NORD = Nordmann fir, TURK = Turkish fir, WHI = white fir.

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Figure 3. Relationship between log-transformed area under the disease progress curve (AUDPC) values and seedling dry weights (expressed as the percentage of the control seedlings) for each host species. Values for all isolates from 15°C and 27°C are combined on the figures to assess overall relationship between root rot severity and AUDPC. Trendlines represent regression between the variables. BAL = balsam fir, CAN = Canaan fir, FRA = Fraser fir, NOB = noble fir, NORD = Nordmann fir, TURK = Turkish fir, WHI = white fir.

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Figure 4. Seed ling root system dry weights (expressed as the percentage of the control seedling weights) among host species for each Phytophthora species and isolate treatment. The 4 Phytophthora species are shown in the vertical facets and the 3 isolates for each species are shown in the horizontal facets. Letters above bars indicate significant differences in root dry weight (% of control) among the 7 Abies spp. for each Phytophthora treatment, as determined by Tukey’s HSD post- hoc analysis. The two

temperatures were analyzed separately, distinguished by upper and lowercase letters.

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Figure 5. Seedling root system dry weights (expressed as the percentage of the control seedling weights) among Phytophthora species for each host species. The 7 Abies species are shown in the vertical facets and the 3 isolates for each Phytophthora treatment are shown in the horizontal facets. Letters above bars indicate significant differences in root dry weights (% of controls) caused by each of the 4 Phytophthora species, as determined by Tukey’s HSD post-hoc analysis. The two temperatures were analyzed separately, distinguished by upper and lowercase letters. Stars above bars indicate treatments in which weights were reduced significantly (p<0.05) from controls.

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Figure 6. Average root rot severity (expressed as the midpoint value of the percentage range) among Phytophthora species for each host species. The 7 Abies species are shown in the vertical facets and the 3 isolates for each Phytophthora treatment are shown in the horizontal facets. Letters above bars indicate significant differences in root rot severity caused by each of the 4 Phytophthora species, as determined by Tukey’s HSD post-hoc analysis. The two temperatures were analyzed separately, distinguished by upper and lowercase letters. Crosses on bars indicate treatments in which % root rot did not differ significantly from controls.

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Figure 7. Average root rot severity (expressed as the midpoint values of the percentage range) among host species for each Phytophthora species and isolate treatment. The 4 Phytophthora species are shown in the vertical facets and the 3 isolates for each species are shown in the horizontal facets. Letters above bars indicate significant differences (p<0.05) in root rot severity between the 7 Abies species, as determined by Tukey’s HSD post-hoc analysis. The two temperatures were analyzed separately, distinguished by upper and lowercase letters.

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Figure 8. Disease progress curves displaying mortality over 16 weeks for 7 Abies species exposed to 3 isolates each of 4 Phytophthora species at two temperatures, 15ºC and 27°C. The x-axis indicates ratings (R1, R2, R3...R8) made biweekly over the course of 16 weeks. Ratings were assessed by estimating the condition of the above-ground foliar parts of each plant on a 1-3 scale where 1 = alive, 2 = failing, 3 = dead. The number of dead trees (ratings of 3) were tallied and averaged across the 5 trees constituting an experimental

unit, as well as across the 5 replicate blocks, to obtain a total % mortality for each treatment at each rating date.

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Figure 9. In vitro radial growth (mm/day) of 3 isolates of each of 4 species of Phytophthora at the two temperatures employed in the greenhouse study. Student’s t-tests were used to assess differences in growth of each isolate for each Phytophthora species between the two temperatures. Statistically significant differences in growth between the two temperatures for a given Phytophthora isolate are indicated by stars above the bars.

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Table 1. Bulk custom soil mix prepared by Specialty Soils (Covington, WA) used to pot seedlings for all experiments.

Quantity Component per yard 2% diatomaceous earth 27.84% Saskatchewan sphagnum peat 17.5% pumice 5% nitrified alder sawdust 50% aged fine bark (hemlock and fir) 0.25 lb. M-ROOTS 3-3-3 (mycorrhizae endo/ecto) 0.013 lb. Treflan 10G 0.38 lb. calcium nitrate (Ca(NO3)2) (15.5-0-0) 0.29 lb. potassium sulfate (K2SO4) (0-0-53) 0.28 lb. Ammophos (NH4H2PO4) (11-52-0) 0.83 lb. ferrous sulfate (FeSO4) (20% Fe) 5.5 lb. Plantacote® (14-9-15) + S (4 mo. release) 0.38 lb. agricultural gypsum (CaSO42H2O) 3.75 lb. limestone flour 6.0 lb. dolomite lime 0.5 oz. manganese sulfate (MnSO4) (2.5% Mn) 0.13 oz. sodium molybdate (Na2MoO4) (2.0% Mo) 1.0 oz. fritted micronutrients 1.07 oz. TriCure media surfactant

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Table 2. Cultures utilized for virulence testing of P. cambivora, P. cinnamomi, P. kelmania, and P. pini isolates. Isolate ID number corresponds indicates how many cultures were obtained for each of the 4 Phytophthora species. Location of origin indicates city & state where isolate was recovered. Host of origin indicates the species of Abies from which each Phytophthora isolate was obtained.

Phytophthora Species Isolate ID Location of Origin Host of Origin NCBI Accession # P. cambivora 01 Stayton, OR A. procera KU053253 P. cambivora 02 Mason Cty., MI A. fraseri N/A P. cambivora 03 Tacoma, WA A. procera KU053263 P. cambivora 04 Puyallup, WA A. procera KU053270 P. cambivora 05 Salem, OR A. procera KU220617 P. cambivora 06 Stayton, OR A. procera KU053251 P. cambivora 07 Maple Valley, WA A. procera KU053259 P. cambivora 08 Eatonville, WA A. procera KU053260 P. cambivora 09 Stayton, OR A. procera KU053252 P. cambivora 10 Enumclaw, WA A. procera KU053264

P. cambivora 11 Canby, OR A. procera KU053255

P. cinnamomi 01 Placerville, CA A. equi-trojani KU053228 97 P. cinnamomi 02 Placerville, CA A. concolor KU053229 P. cinnamomi 03 Placerville, CA A. nordmanniana KU053227 P. cinnamomi 04 Camino, CA A. nordmanniana KU053226 P. cinnamomi 05 Raleigh, NC A. fraseri N/A P. cinnamomi 06 Raleigh, NC A. bornmuelleriana KU220613 P. cinnamomi 07 Raleigh, NC A. equi-trojani KU220612 P. kelmania 01 Litchfield, CT A. fraseri KU053233 P. kelmania 02 Groton, NY A. fraseri KU053242 P. kelmania 03 Groton, NY A. fraseri KU220615 P. kelmania 04 Groton, NY A. fraseri KU053244 P. kelmania 05 Manitowac Cty., WI A. balsamea var. phanerolepis N/A P. kelmania 06 Jefferson, NC A. bornmuelleriana KU053247 P. kelmania 07 Jefferson, NC A. fraseri KU053249 P. pini 01 Warren, CT A. balsamea var. phanerolepis KU053230 P. pini 02 Groton, NY A. fraseri KU053241 P. pini 03 Allegan Cty., MI A. fraseri AY995350 P. pini 04 Jefferson, NC A. fraseri KU053248

Table 3. Rho and p values from Pearson’s Product Moment Correlation examine agreement of the 3 response variables assessed in this study (area under the disease progress curve (AUDPC); root rot rating (midpoint value of the percentage range); and dry weights (% of the control seedlings) for each of the 7 host species (left panel). Results of multiple linear regression analysis to understand the predictive power of using root rot rating and dry weights to forecast AUDPC for each host species (right panel).

Pearson’s Product Moment Correlation Multiple Linear Regression

AUDPC AUDPC Rot Rating AUDPC v. v. v. v. Rot Rating Dry Weight Dry Weight Rot Rating & Dry Weight R2 = 0.72 r = 0.97 r = -0.74 r = -0.81 p (Rot) = 0.0002 noble p < 0.0001 p < 0.0001 p < 0.0001 p (Wt.) = 0.6326

R2 = 0.79 r = 0.95 r = -0.63 r = -0.79 p (Rot) = 0.0004 Fraser p < 0.0001 p = 0.0005 p < 0.0001 p (Wt.) = 0.0084

R2 = 0.94 r = 0.86 r = -0.50 r = -0.74 p (Rot) < 0.0001 white p < 0.0001 p = 0.009 p < 0.0001 p (Wt.) = 0.0014

R2 = 0.95 r = 0.86 r = -0.72 r = -0.80 p (Rot) < 0.0001 balsam p < 0.0001 p < 0.0001 p < 0.0001 p (Wt.) = 0.039

R2 = 0.92 r = 0.86 r = -0.82 r = -0.74 p (Rot) < 0.0001 Canaan p < 0.0001 p < 0.0001 p < 0.0001 p (Wt.) = 0.0034

R2 = 0.87 r = 0.95 r = -0.83 r = -0.94 p (Rot) < 0.0001 Nordmann p < 0.0001 p < 0.0001 p < 0.0001 p (Wt.) = 0.054

R2 = 0.76 r = 0.92 r = -0.80 r = -0.76 p (Rot) < 0.0001 Turkish p < 0.0001 p < 0.0001 p < 0.0001 p (Wt.) = 0.0608

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Table 4. Average percent mortality of 7 Abies spp. after 16 weeks exposure to 3 isolates each of 4 Phytophthora species at two temperatures. Percentages represent totals from all 5 blocks for each treatment. Analyses of variance were performed using the PROC MIXED procedure with post-hoc analysis using Tukey’s HSD to determine mean separations between fir species.

15°C 27°C Treatment Treatment

P. cambivora P. cinnamomi P. kelmania P. pini P. cambivora P. cinnamomi P. kelmania P. pini 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Fir spp. noble 28a 44a 60a 16a 72ab 100a 52a 20a 8a 40a 64a 68a 80a 92a 96a 88a 100a 100a 92a 100a 96a 96a 92a 96a Fraser 20b 24a 32b 8a 76a 80b 40ab 12a 20a 32ab 76a 68a 32b 64b 56b 88a 100a 100a 100a 96a 92a 88ab 96a 100a white 8b 4b 28b 4a 44bcd 64bc 12ab 4a 0a 20b 12b 28b 16bc 12c 28c 12c 76c 88b 56b 52b 40b 64b 60b 64b

balsam 0b 0b 0b 4a 52bc 40cd 4b 0a 0a 8b 0b 4b 8bc 8c 0c 48b 92ab 100a 68ab 84a 32b 28c 32bc 48b

b b b a cd cd b a a b b b c c c bc b bc bc bc bc c c c Canaan 0 0 0 0 12 16 0 0 0 8 0 0 0 0 0 44 84 80 36 24 16 16 4 16 99 Nordmann 0b 0b 0b 0a 8d 28cd 0b 0a 0a 0b 0b 0b 0c 0c 0c 12c 56c 70bc 0c 0c 0c 0c 0c 0c Turkish 0b 0b 0b 0a 4d 8d 0b 0a 0a 0b 0b 0b 0c 0c 0c 4c 52c 56c 0c 4c 4c 0c 4c 0c

a Letters next to measurements indicate significantly different mean mortality among the 7 host species within each Phytophthora × isolate treatment

APPENDIX: Chapter 3 Figures and Tables.

Figure 1. Percent mortality of 36 seed sources of Turkish fir, 2 seed sources of Nordmann fir, and 1 seed source each of noble and Fraser firs. Proportions were calculated by dividing pooled 2-year totals of dead trees from each seed source by the total number of trees per source that were employed in two years of the field study (N=60 trees). Three distinct provenances of Turkish fir (Bolu, Karabük, and Adapazarı) were represented by 12 seed sources each, as indicated near the top of the graph. Within each provenance, 4 seed sources represented mother trees growing at 3 distinct elevation categories (E1 = 1,030-1,200 m; E2 = 1,230-1,400 m; E3 = 1,430-1,700 m).

BOLU KARABÜK ADAPAZARI 100 E3 E2 E1 E1 E2 E3

E1 E2 E3 90

80

70 100 60 50 40 30 20

% Mortality, 14 wks. post inoculation post wks. 14 Mortality, % 10

0

2 4 5 7 8

33 12 13 14 15 16 18 19 21 23 24 25 28 29 31 36 38 39 40 41 47 48 50 51 52 54 55 57 58 59 60

noble

Fraser

Nord 1 Nord 2 Nord Seed Source

Figure 2. Percent mortality among Turkish fir produced from three provenances. The 2-year totals of dead trees from the 12 seed sources that represented a provenance were tallied and divided by the total number of trees of each provenance employed in two years of the field study (N=720 trees per provenance). Letters above the bar indicate significance of the differences in mortality from each provenance as calculated in the post-hoc Dunn’s and Nemenyi’s tests to analyze sample pairs for stochastic dominance. Error bars represent standard deviation.

ab

b

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Figure 3. Mortality of seedlings from seed sources from 3 elevation categories within Bolu, Karabük, or Adapazarı Turkish fir provenances. Separate one-way ANOVAs of the pooled 2- year mortality data were calculated to assess statistical differences in total seedling mortality between elevation categories within each provenance. A significant ANOVA for the Adapazarı provenance prompted pairwise comparisons using Fisher’s Least Squared Difference (LSD) test. LSD analysis indicated that the lowest elevation (1,130-1,200 m) differed significantly from the moderate and high elevation categories; denoted by a star. Error bars indicate standard error.

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Figure 4. Percentage of the two-year total seedlings of each Abies host killed by each Phytophthora spp. The total number of dead seedlings (N) varies for each host, and is presented in the legend. Each provenance of Turkish fir is represented separately.

Nordmann N=8 trees Fraser N=51 trees Turkish (Karabuk) N=14 trees

noble N=45 trees Turkish (Adapazari) N=39 trees Turkish (Bolu) N=49 trees 100 90 80 70 60

50

40 103

30 Percent Mortality Percent 20 10 0 P. cryptogea P. cinnamomi P. cactorum P. cambivora P. pini

Phytophthora Species

Figure 5. Percentage of 2-year total tree mortality caused by each Phytophthora species, irrespective of host species. Values were determined by tallying the total number of times each Phytophthora spp. was isolated and dividing by the total number of dead seedlings. Columns with different letters are significantly different based on a significant ANOVA (p<0.001) and post-hoc analysis using Fisher’s LSD.

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Figure 6. Map depicting the southwestern Black Sea and Marmara regions of northwestern Turkey showing the location of the three Turkish fir provenances (Adapazarı, Bolu, and Karabük) assessed by this study.

Table 1. Phytophthora cultures utilized in both years of the field experiment.

Phytophthora spp. WSU Culture Coll. ID* Host of Origin Geographic origin P. cactorum MM60-1192 Malus spp. Wenatchee, WA P. cambivora WA-CC-12 No-s4 Abies procera Maple Valley, WA P. cinnamomi M10-0040 Arbutus menziesii Florence, OR P. cryptogea 109-004 Abies procera Puyallup, WA P. pini 109-0030 Abies procera Puyallup, WA *Internal culture collection housed at Washington State University, Puyallup Research and Extension Center, 2606 W. Pioneer, Puyallup, WA 98371.

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